Jekyll2023-05-10T22:32:18+00:00https://sarahtanja.github.io/lab-book/feed.xmlSarah’s Lab NotebookAn open lab notebook where I can save and share protocols, posts, and data from my Masters thesis research projectsSarah S.D. Tanjamake-blog2023-05-09T00:00:00+00:002023-05-09T00:00:00+00:00https://sarahtanja.github.io/lab-book/blog/goals/committee%20meetings/protocols/sample%20processing/bioinformatic%20analyses/make-blog<ol>
<li>Use the <a href="https://github.com/mmistakes/mm-github-pages-starter/generate">Minimal Mistakes remote theme starter</a> to generate a new repository. Give this new repository a</li>
<li>Go To <a href="https://mmistakes.github.io/minimal-mistakes/docs/quick-start-guide/#remote-theme-method">QUICK START GUIDE</a> and follow instructions for Remote Theme Method</li>
</ol>
<h4 id="embed-image">Embed Image</h4>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/qbit-rna-iq.png" alt="qbit " /><em>infographic sourced from <a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Fqubit-rna-iq-assay-fluorometric-rna-quality-assessment-poster.pdf">Thermo Fisher</a></em></p>
<h4 id="wrap-notice">Wrap Notice</h4>
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<p class="notice--danger"><strong>Danger Notice:</strong> Lorem ipsum dolor sit amet, <a href="#">consectetur adipiscing</a> elit. Integer nec odio. Praesent libero. Sed cursus ante dapibus diam. Sed nisi. Nulla quis sem at nibh elementum imperdiet.</p>
<p class="notice--success"><strong>Success Notice:</strong> Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer nec odio. Praesent libero. Sed cursus ante dapibus diam. Sed nisi. Nulla quis sem at <a href="#">nibh elementum</a> imperdiet.</p>Sarah S.D. TanjaUse the Minimal Mistakes remote theme starter to generate a new repository. Give this new repository a Go To QUICK START GUIDE and follow instructions for Remote Theme MethodApril ‘23 Goals2023-04-05T00:00:00+00:002023-04-05T00:00:00+00:00https://sarahtanja.github.io/lab-book/goals/goals-APR23<h1 id="april-23-goals">April ‘23 Goals</h1>
<h2 id="primary-focus">Primary Focus</h2>
<h3 id="dnarna-extractions--qubit">DNA/RNA Extractions & Qubit!</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Currently waiting to receive the ‘Mortexer’ … can’t do extractions until we get that instrument!</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1st round:
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-Ea</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />2-Ea</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />2nd round:
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-CAa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-CHa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-LAa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-LHa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-HAa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-HHa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-OAa</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />1-OHa</li>
</ul>
</li>
</ul>
<hr />
<h3 id="sequencing-quotes-results">Sequencing Quotes Results</h3>
<blockquote>
<p>Quotes are for library prep & sequencing</p>
</blockquote>
<p>16S</p>
<ul>
<li>data format - raw FASTQ files</li>
</ul>
<p>RNA-seq</p>
<ul>
<li>
<p>The size of the Montipora capitata genome is 614.5 million base pairs.</p>
</li>
<li>
<p>Illumina, 2x150bp (for gene expression profiling)</p>
</li>
<li>
<p>poly-A RNA selection</p>
</li>
<li>
<p>15-20 million raw paired-end reads per sample</p>
</li>
<li>
<p>data format - raw FASTQ files</p>
</li>
</ul>
<hr />
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://nwgc.gs.washington.edu/">UW Northwest Genomics Center</a> (email sent 05APR2023)</p>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.azenta.com/">Azenta</a> Life Sciences (previously Genewiz)
<ul>
<li>
<p>sequencing platform:</p>
</li>
<li>
<p>RNA-seq, 10 samples: 23-25 business days, 15-20 million reads per sample, PolyA selection for mRNA, ERCC spike-in, $180 per sample with promo, $1800 total pre-tax</p>
</li>
<li>
<p>16S, 10 samples: 20ng/uL DNA concentration, 25uL volume, $95 per sample, $950 total pre-tax</p>
</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://wikis.utexas.edu/display/GSAF/Home+Page">Univ. of Texas Austin Genomic Sequencing Analysis Facility (GSAF)</a>
<ul>
<li>
<p>sequencing platform: <a href="https://www.illumina.com/systems/sequencing-platforms/miseq/specifications.html">MiSeq V2</a></p>
</li>
<li>
<p>GSAF uses BaseSpace for NGS Illumina data delivery, If you do not have an account, please sign up for one <a href="https://login.illumina.com/platform-services-manager/?rURL=https://basespace.illumina.com&clientId=basespace&clientVars=aHR0cHM6Ly9iYXNlc3BhY2UuaWxsdW1pbmEuY29tL2Rhc2hib2FyZA&redirectMethod=GET#/register">here</a>.</p>
</li>
<li>
<p>$260.39 per sample RNA-seq library prep with poly(A) enrichment</p>
</li>
<li>
<p>RNA-seq, 10 samples, $32,642.71🤯</p>
</li>
<li>
<p><strong>Tag-seq, 20 samples $1,500 ($75 per sample, must be sent in 96-well plate)</strong></p>
</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.mrdnalab.com/contact.html">Mr. DNA Lab</a> (email sent 05APR2023)</p>
<ul>
<li>
<p>RNA-seq, $580 per sample</p>
</li>
<li>
<p>16S, $75 per sample + $100 library prep fee</p>
</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://fyrdiagnostics.com/">FYR Diagnostics</a> (email sent 05APR2023)
<ul>
<li>They do not do RNA depletion/poly(A) enrichment… instead they use a <a href="https://watchmakergenomics.com/portfolio/rna-solutions/">Watchmaker genomics library prep</a> for target enrichment applications</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://gc3f.uoregon.edu/sample-prep-services#Illumina_prices">University of Oregon Genomics Center</a>
<ul>
<li>
<p>RNA-seq, $255 per sample, (does not include poly(A) enrichment! 1 to 100ng of purified total, polyA enriched RNA in 10uL of RNase-free water or buffer</p>
</li>
<li>
<p>16S v3-v4 region, $1,400 per plate of 95 samples, any amount of pure DNA (more = better) in at least 30uL volume</p>
</li>
</ul>
</li>
</ul>
<h3 id="send-off-samples-for-sequencing-service">Send off Samples for Sequencing Service</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />pick quote provider</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />send for sequencing ( 2-4 weeks )</li>
</ul>
<h3 id="fish-546-in-sp23">FISH 546 in SP23</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />prepare scripts to do RNA-seq differential gene expression analysis in-class</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />use ‘fake data’ from publicly available <em>M. capitata</em> RNA-seq data</li>
</ul>
<h2 id="supporting-roles">Supporting Roles</h2>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing coordinating for GSIS speaker Dr. Nicola S. Smith</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing participation in Student Advisory Council</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing mentoring with <a href="https://github.com/norahess/ibis">IBIS mentee Nora Hessen</a></p>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />laboratory work! do DNA/RNA extraction together</li>
</ul>
</li>
</ul>
<h2 id="looking-ahead">Looking Ahead…</h2>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/gantt-timeline.png" alt="gantt-timeline" /></p>
<h4 id="spring-2023">Spring 2023</h4>
<p>May</p>
<ul>
<li>
<p>analyze sequences with Nora for IBIS project (QA/QC… both run same analysis and see if we get same result?)</p>
</li>
<li>Celebrate IBIS mentee project completion</li>
<li>assess how we proceed with extraction & sequencing (did we see something interesting to warrant paying for more sequences? do we need to think about finding additional funding to pay for more sequencing?)</li>
<li>continue bioinformatics pipelines</li>
</ul>
<h4 id="summer-2023">Summer 2023</h4>
<p>June</p>
<ul>
<li>June 9th: Spring Quarter ends</li>
<li>June 10th: FHL Marine Botany TAship starts at Friday Harbor Labs!</li>
<li>begin drafting manuscript(s)</li>
</ul>
<p>July</p>
<ul>
<li>July 15th: FHL Marine Botany TAship ends!</li>
<li>continue drafting manuscript</li>
<li>continue bioinformatics pipelines</li>
</ul>
<p>Aug</p>
<ul>
<li>continue drafting manuscript</li>
<li>continue bioinformatics pipelines</li>
</ul>Sarah S.D. TanjaApril ‘23 GoalsQubit RNA Broad Range Protocol2023-03-20T00:00:00+00:002023-03-20T00:00:00+00:00https://sarahtanja.github.io/lab-book/protocols/protocol-qbit<p>The aim of this protocol is to take the extracted RNA from the <a href="https://sarahtanja.github.io/lab-book/protocols/lab/protocol-DNARNAextraction/">Dual DNA & RNA Extraction Protocol</a> step and check it for <strong>integrity & quality</strong>. This is a necessary quality check (QC) because we want to make sure the samples we send for sequencing actually have enough good RNA in them to sequence.</p>
<p>The Qubit allows us to measure RNA integrity & quality (IQ) extracted in our samples. For high-quality RNA, we’re looking for long and intact strands of RNA.</p>
<h2 id="background">Background</h2>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/qbit-rna-iq.png" alt="qbit " /><em>infographic sourced from <a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Fqubit-rna-iq-assay-fluorometric-rna-quality-assessment-poster.pdf">Thermo Fisher</a></em></p>
<p>👀<a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FQubit_RNA_BR_Assay_UG.pdf">Qubit RNA Broad Range (BR) Assay Kit Manual</a></p>
<p>👀<a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FQRC10210.pdf">Qubit RNA Broad Range (BR) Assay Kit Quick Reference</a></p>
<h2 id="materials-list">Materials List</h2>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.thermofisher.com/order/catalog/product/Q33238?SID=srch-srp-Q33238">Qubit Fluorometer</a></p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.thermofisher.com/order/catalog/product/Q10210">Qubit Broad Range Assay Kit</a></p>
<p class="notice--warning"><strong>Warning Notice:</strong> Standards must be stored in a refrigerator! Kits are stable for 6 months</p>
<p class="notice--danger"><strong>Danger Notice:</strong> Kit contains DMSO 67-68-5 in the RNA dye Reagent 200X concentrate, a combustible liquid that is a mild skin and eye irritant and may be absorbed into the body through the skin. If accidental contact with skin or eye happens, rinse with plenty of water, immediate medical attention is not required (according to the <a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2FSDS%2FQ10210COMPONENTA_MTR-NALT_EN.pdf">SDS</a>)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.thermofisher.com/order/catalog/product/Q32856?SID=srch-srp-Q32856">Qubit Assay Tubes</a></p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />15mL Falcon tube (to prepare working solution) 🧪</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />0.5mL PCR tube rack</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P2 pipette + tips</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P10 pipette + tips</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P1000 pipette + tips</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />nitrile gloves 🧤</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />safety glasses 👓</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />vortexer</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />USB thumb-drive</p>
</li>
</ul>
<h2 id="protocol">Protocol</h2>
<h3 id="quick-reference-overview">Quick-Reference Overview</h3>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/qbit-quick.png" alt="qbit " /><em>infographic sourced from <a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FQRC10210.pdf">Qubit RNA Broad Range (BR) Assay Kit Quick Reference</a></em></p>
<h3 id="advanced-prep">Advanced Prep</h3>
<p>Thaw samples, or aliquots of sample, on ice. Only 1uL is needed from the sample to run Qubit. This tiny amount is prone to pipetting error, which can be influenced by temperature. Make sure the aliquot you are pulling 1uL from is well thawed.</p>
<h3 id="set-up-2-assay-tubes-for-the-standards-and-1-tube-for-each-sample">Set up 2 assay tubes for the standards and 1 tube for each sample</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />label assay tube lids</li>
</ul>
<p class="notice--warning"><strong>Warning Notice:</strong> Label only the cap of the assay tube, the sides need to remain clear for the fluorometer to penetrate through and read the sample</p>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />rip off sheet of foil to cover sample tube rack to protect from light</li>
</ul>
<p class="notice--warning"><strong>Warning Notice:</strong> Qubit RNA BR dye-reagent (component A) is light sensitive, and must be kept in the dark (tube wrapped in foil)</p>
<h3 id="step-2-prepare-working-solution">Step 2. Prepare Working Solution</h3>
<p class="notice--success"><strong>tldr:</strong> For 8 samples and 2 standards, you will need 2mL of working solution, to prepare it in a 1:200 dilution of dye-reagent to buffer, pipette 10uL of Qubit dye-reagent plus 1,990uL of Qubit buffer into a 15mL falcon tube, cap & invert to mix.</p>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />calculate volume of working solution you need –> <strong>( 2 x 190uL ) + (no. of samples x 199uL)</strong> <em>(ex. 2 x 190uL + 8 x 199uL) = 1,972uL round up to 2ml</em></p>
<blockquote>
<p>standard #1 - 190uL working solution, 10uL standard</p>
<p>standard #2 - 190uL working solution, 10uL standard</p>
<p>sample 1 - 199uL working solution, 1uL sample</p>
<p>sample 2 - 199uL working solution, 1uL sample</p>
<p>sample 3 - 199uL working solution, 1uL sample</p>
<p>.. etc.</p>
</blockquote>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />The working solution is 1 part dye-reagent to 200 parts buffer</p>
<ul>
<li>
<p>calculate a 1:200 dilution of dye-reagent to buffer</p>
</li>
<li>divide working solution volume (uL) by 200 to find volume (uL) of dye-reagent <em>(ex. 2000uL / 200 = 10uL dye-reagent)</em></li>
<li>subtract volume (uL) of dye-reagent from working solution volume to get buffer volume. <em>(ex. 2000uL - 10uL = 1990uL buffer)</em></li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />take 15mL falcon tube, add above volumes of dye-reagent and buffer to make the final working solution volume. Cap & invert falcon tube to mix.</p>
<p class="notice--info"><strong>Note:</strong> The final volume in each tube must be 200 µL. Each standard tube requires 190 µL of Qubit™ working solution, and each sample tube requires anywhere from 180–199 µL. Ensure that you have sufficient Qubit™ working solution to accommodate all standards and samples, with a little ‘extra’.</p>
</li>
</ul>
<h3 id="step-3-pipette-working-solution-standards--samples-into-assay-tubes">Step 3. Pipette Working Solution, Standards, & Samples into assay tubes</h3>
<h4 id="standard-tubes">Standard tubes</h4>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Add 190uL of working solution to each standard tube</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Add 10uL of standard (#1 & #2) to each correspondingly labelled tube</li>
</ul>
<h4 id="sample-tubes">Sample tubes</h4>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Add 199 uL of working solution into each sample tube</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Add 1uL of sample into each correspondingly labelled tube (P2 pipette)</li>
</ul>
<p class="notice--warning"><strong>Warning:</strong> temperature affects pipetting accuracy, which is very important when pipetting tiny amounts (1uL!!), so make sure sample is on ice, but well-thawed.</p>
<h3 id="step-4-vortex-standards-and-samples-for-23-seconds-and-let-sit-at-room-temperature-for-2-minutes">Step 4. Vortex standards and samples for 2–3 seconds and let sit at room temperature for 2 minutes</h3>
<h3 id="step-5-read-samples">Step 5. Read Samples</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Select RNA Broad Range Assay on the Qubit® 2.0 Fluorometer to calibrate with standards</li>
</ul>
<p class="notice--warning"><strong>Warning Notice:</strong> Qubit™ assays delivers optimal performance when all solutions are at room temperature; temperature fluctuations can influence the accuracy of the assay. To minimize temperature fluctuations, insert all assay tubes into the Qubit™ Fluorometer only for as much time as it takes for the instrument to measure the fluorescence; the Qubit™ Fluorometer can raise the temperature of the assay solution significantly, even over a period of a few minutes. Do not hold the assay tubes in your hand before reading because this warms the solution and results in a low
reading.</p>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />After calibration, read the samples with the same RNA Broad Range Assay selection on the Qubit Fluorometer. Cross your fingers they are in desired range 🤞</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Record RNA IQ values</p>
<blockquote>
<hr />
<table>
<thead>
<tr>
<th>sample</th>
<th>qubit-rna-iq</th>
<th>qubit-run-date</th>
</tr>
</thead>
<tbody>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
</blockquote>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Download RNA IQ values using USB thumb-drive</p>
</li>
</ul>Sarah S.D. TanjaThe aim of this protocol is to take the extracted RNA from the Dual DNA & RNA Extraction Protocol step and check it for integrity & quality. This is a necessary quality check (QC) because we want to make sure the samples we send for sequencing actually have enough good RNA in them to sequence.March ‘23 Goals2023-02-27T00:00:00+00:002023-02-27T00:00:00+00:00https://sarahtanja.github.io/lab-book/goals/goals-MAR23<h1 id="march-23-goals">March ‘23 Goals</h1>
<h2 id="primary-focus">Primary Focus</h2>
<h3 id="dnarna-extraction-protocols">DNA/RNA Extraction Protocols</h3>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Research DNA/RNA extraction kits (compete on 05FEB23)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Buy Quick-DNA/RNA Miniprep Plus Kit , 10 preps ($106.70, requested 08FEB23 on Hall Grant Budget)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Receive kits (complete on 23FEB23)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Draft <a href="https://sarahtanja.github.io/lab-book/protocols/lab/protocol-DNARNAextraction/">protocol</a> (complete on 15FEB23 )</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Locate (… or buy or borrow) 300uL/500uL & 1000uL pipettes & tips</p>
<ul>
<li>pipettes serviced by gilson rep end of Feb ‘23</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Protocol dry-run walk-thru with IBIS mentee</p>
<ul>
<li>
<p>dry-run walk-thru on 30MAR23</p>
</li>
<li>
<p>samples located in ‘sucks’ -80 freezer, top shelf, far back right corner</p>
</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Edit/amend <a href="https://sarahtanja.github.io/lab-book/protocols/lab/protocol-DNARNAextraction/">protocol</a></p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Trial methods with subset of samples <strong>Note:</strong> <em>We originally thought about using Callum’s coral fragments for the trial, but these were not held at -80C, and therefore the RNA step would not work properly</em></p>
</li>
</ul>
<h3 id="get-sequencing-quotes">Get Sequencing Quotes</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Get general idea of costs ( $80/sample for 16S & $200/sample for RNAseq )</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Answer/identify common quote questions:
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />What type of library prep is needed?</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />What is the minimum number of reads per sample?</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />How many samples to sequence?</p>
<ul>
<li>
<p>start with a practice of 2</p>
</li>
<li>follow-up with 8 more (total of 10 preps in kit)</li>
<li>buy 50-prep kit and continue if extractions went well</li>
</ul>
</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />UW Northwest Genomics Center</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Azenta Life Sciences (previously Genewiz)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Univ. of Texas</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Mr. DNA Lab</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />FYR Diagnostics (20% off promo)</li>
</ul>
<h3 id="send-off-samples-for-sequencing-service">Send off Samples for Sequencing Service</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />finalize extraction strategy (how many samples, from which treatments)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />pick quote provider</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />extract DNA/RNA from samples <strong>Note:</strong> <em>this will be a subset of samples, likely the control vs. highest treatments</em></li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />send for sequencing ( 2-4 weeks )</li>
</ul>
<h3 id="start-fish-546-in-sp23">Start FISH 546 in SP23</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />setup Linux Subsystem for Windows in preparation of Bioinformatics Course</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />receive sequences and prepare to do analysis in-class</li>
</ul>
<h2 id="supporting-roles">Supporting Roles</h2>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Complete SMEA/FISH 539 coursework WI23</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Start FISH 546 coursework SP23</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing coordinating for GSIS speaker Dr. Nicola S. Smith</p>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing participation in Student Advisory Council
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />CoEnv Assistant Dean of Diversity Equity & Inclusion (ADDEI) hire student input</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing mentoring with <a href="https://github.com/norahess/ibis">IBIS mentee Nora Hessen</a>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />brainstorming & meeting every Wednesday for 2hrs</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />laboratory work! do DNA/RNA extraction together</p>
</li>
</ul>
</li>
</ul>
<h2 id="looking-ahead">Looking Ahead…</h2>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/gantt-timeline.png" alt="gantt-timeline" /></p>
<h4 id="spring-2023">Spring 2023</h4>
<p>April</p>
<ul>
<li>receive trial sequences</li>
<li>analyze the sequences in Bioinformatics course</li>
<li>2nd round DNA/RNA extraction, & send for sequencing</li>
</ul>
<p>May</p>
<ul>
<li>
<p>analyze sequences with Nora for IBIS project (QA/QC… both run same analysis and see if we get same result?)</p>
</li>
<li>Celebrate IBIS mentee project completion</li>
<li>assess how we proceed with extraction & sequencing (did we see something interesting to warrant paying for more sequences? do we need to think about finding additional funding to pay for more sequencing?)</li>
<li>continue bioinformatics pipelines</li>
</ul>
<h4 id="summer-2023">Summer 2023</h4>
<p>June</p>
<ul>
<li>June 9th Spring Quarter ends</li>
<li>June 20th Summer A term begins</li>
<li>FHL Marine Botany TAship?</li>
<li>begin drafting manuscript(s)</li>
</ul>
<p>July</p>
<ul>
<li>July 19th Summer A term ends</li>
<li>continue drafting manuscript</li>
<li>continue bioinformatics pipelines</li>
</ul>
<p>Aug</p>
<ul>
<li>continue drafting manuscript</li>
<li>continue bioinformatics pipelines</li>
</ul>
<h2 id="advisor-recommendations">Advisor Recommendations</h2>Sarah S.D. TanjaMarch ‘23 GoalsFeb ‘23 Goals2023-02-08T00:00:00+00:002023-02-08T00:00:00+00:00https://sarahtanja.github.io/lab-book/goals/goals-FEB23<h1 id="february-23-goals">February ‘23 Goals</h1>
<h2 id="primary-focus">Primary Focus</h2>
<h3 id="conduct-1st-committee-meeting">Conduct 1st Committee Meeting</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Write committee agreements (complete on 01FEB23)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Build ‘committee presentation’ blog post (<a href="https://sarahtanja.github.io/lab-book/committee%20meetings/committee-no1/">here</a>) (complete on 05FEB23)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Host meeting (complete on 06FEB23)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Integrate feedback into <a href="https://sarahtanja.github.io/lab-book/committee%20meetings/committee-no1/#feedback">master’s thesis committee meeting #</a>1 blog (complete on 07FEB23)</li>
</ul>
<h3 id="dnarna-extraction-protocols">DNA/RNA Extraction Protocols</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Research DNA/RNA extraction kits (compete on 05FEB23)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Buy Quick-DNA/RNA Miniprep Plus Kit , 10 preps ($106.70, requested 08FEB23 on Hall Grant Budget)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Receive kits (complete on 23FEB23)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Draft <a href="https://sarahtanja.github.io/lab-book/protocols/lab/protocol-DNARNAextraction/">protocol</a> (complete on 15FEB23 )</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Locate (… or buy or borrow) 300uL/500uL & 1000uL pipettes & tips</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Protocol dry-run walk-thru with IBIS mentee</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Edit/amend <a href="https://sarahtanja.github.io/lab-book/protocols/lab/protocol-DNARNAextraction/">protocol</a></li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Trial methods with subset of samples <strong>Note:</strong> <em>We originally thought about using Callum’s coral fragments for the trial, but these were not held at -80C, and therefore the RNA step would not work properly</em></li>
</ul>
<h3 id="get-sequencing-quotes">Get Sequencing Quotes</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Get general idea of costs ( $80/sample for 16S & $200/sample for RNAseq )</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Answer/identify common quote questions:
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />What type of library prep is needed?</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />What is the minimum number of reads per sample?</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />How many samples to sequence?</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />UW Northwest Genomics Center</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Azenta Life Sciences (previously Genewiz)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Univ. of Texas</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Mr. DNA Lab</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />FYR Diagnostics (20% off promo)</li>
</ul>
<h2 id="supporting-roles">Supporting Roles</h2>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Complete SMEA/FISH 539 coursework</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing coordinating for GSIS speaker Dr. Nicola S. Smith</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing participation in Student Advisory Council
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />CoEnv Strategic Planning student working group</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />CoEnv Assistant Dean of Diversity Equity & Inclusion (ADDEI) hire student input</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Ongoing mentoring with <a href="https://github.com/norahess/ibis">IBIS mentee Nora Hessen</a>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />develop project (DNA/RNA extraction & alpha & beta diversity analysis!)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />lab training & certificates</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />brainstorming & meeting every Wednesday for 2hrs</li>
</ul>
</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Apply for FHL Marine Botany TAship</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" checked="checked" />Apply for WSG Grad Student Assistant, locally sourced seafood project</li>
</ul>
<h3 id="apply-for-pcc-climate-solutions-project-funding">Apply for <a href="https://pcc.uw.edu/research/funding-opportunities/">PCC Climate Solutions Project Funding</a></h3>
<p>$5k max for solutions-based projects, capstone programs (IBIS), outreach efforts with community organizations and partners. Can be used for Financial Support, Training, Publication & Travel. Rolling application. Would like to apply for this to be used to help IBIS mentee, via more sequencing or publication? Need to brainstorm how to angle ‘solutions-based’ project.</p>
<h2 id="looking-ahead">Looking Ahead…</h2>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/gantt-timeline.png" alt="gantt-timeline" /></p>
<h4 id="spring-2023">Spring 2023</h4>
<p>March</p>
<ul>
<li>pick a sequening service</li>
<li>finalize extraction strategy (how many samples, from which treatments)</li>
<li>extract DNA/RNA from samples <strong>Note:</strong> <em>this will be a subset of samples, likely the control vs. highest treatments</em></li>
<li>send for sequencing ( 2-4 weeks )</li>
<li>setup Linux Subsystem for Windows in preparation of Bioinformatics Course</li>
<li>March 27 Spring Quarter begins : Dr. Robert’s Bioinformatics Course</li>
</ul>
<p>April</p>
<ul>
<li>receive trial sequences</li>
<li>analyze the sequences in Bioinformatics course</li>
<li>2nd round DNA/RNA extraction, & send for sequencing</li>
</ul>
<p>May</p>
<ul>
<li>
<p>analyze sequences with Nora for IBIS project (QA/QC… both run same analysis and see if we get same result?)</p>
</li>
<li>Celebrate IBIS mentee project completion</li>
<li>assess how we proceed with extraction & sequencing (did we see something interesting to warrant paying for more sequences? do we need to think about finding additional funding to pay for more sequencing?)</li>
<li>continue bioinformatics pipelines</li>
</ul>
<h4 id="summer-2023">Summer 2023</h4>
<p>June</p>
<ul>
<li>June 9th Spring Quarter ends</li>
<li>June 20th Summer A term begins</li>
<li>FHL Marine Botany TAship?</li>
<li>begin drafting manuscript(s)</li>
</ul>
<p>July</p>
<ul>
<li>July 19th Summer A term ends</li>
<li>continue drafting manuscript</li>
<li>continue bioinformatics pipelines</li>
</ul>
<p>Aug</p>
<ul>
<li>continue drafting manuscript</li>
<li>continue bioinformatics pipelines</li>
</ul>
<h2 id="advisor-recommendations">Advisor Recommendations</h2>Sarah S.D. TanjaFebruary ‘23 GoalsRendering Images in Jekyll Minimal Mistakes2023-02-02T00:00:00+00:002023-02-02T00:00:00+00:00https://sarahtanja.github.io/lab-book/blog/post-image-troubleshoot<p>This post is for troubleshooting why my images are not rendering on the Minimal Mistakes site.</p>
<p>The minimal mistakes jekyll <a href="https://github.com/mmistakes/minimal-mistakes/tree/master/docs/assets/images">assets/images</a> folder images are all either .png or .jpg files</p>
<p>The <a href="https://mmistakes.github.io/minimal-mistakes/post%20formats/post-image-standard/">post</a> & <a href="https://github.com/mmistakes/minimal-mistakes/blob/master/docs/_posts/2010-08-05-post-image-standard.md">file</a> on including a standard image says:</p>
<p>The preferred way of using images is placing them in the <code class="language-plaintext highlighter-rouge">/assets/images/</code> directory and referencing them with an absolute path. Prepending the filename with <code class="language-plaintext highlighter-rouge">{{ site.url }}{{ site.baseurl }}/assets/images/</code> will make sure your images display properly in feeds and such.</p>
<p>In my <code class="language-plaintext highlighter-rouge">config.yml</code>file I added the lines:</p>
<div class="language-yml highlighter-rouge"><div class="highlight"><pre class="highlight"><code><span class="na">url </span><span class="pi">:</span> <span class="s2">"</span><span class="s">sarahtanja.github.io"</span>
<span class="na">baseurl </span><span class="pi">:</span> <span class="s2">"</span><span class="s">/lab-book"</span>
</code></pre></div></div>
<p><strong>HTML:</strong></p>
<div class="language-html highlighter-rouge"><div class="highlight"><pre class="highlight"><code><span class="nt"><img</span> <span class="na">src=</span><span class="s">"{{ site.url }}{{ site.baseurl }}/assets/images/githuboctocat.svg"</span> <span class="na">alt=</span><span class="s">""</span><span class="nt">></span>
</code></pre></div></div>
<p><img src="{{ site.url }}{{ site.baseurl }}/assets/images/guthuboctocat.svg" alt="" /></p>
<p>… nothing</p>
<p><strong>Kramdown:</strong></p>
<div class="language-markdown highlighter-rouge"><div class="highlight"><pre class="highlight"><code><span class="p">![</span><span class="nv">alt</span><span class="p">](</span><span class="sx">{{</span> site.url }}{{ site.baseurl }}/assets/images/githuboctocat.svg)
</code></pre></div></div>
<p><img src="{{ site.url }}{{ site.baseurl }}/assets/images/githuboctocat.svg" alt="alt" /></p>
<p>…</p>
<p><strong>Kramdown without ‘ wrap’:</strong></p>
<pre><code class="language-kramdown">![img-png](https://sarahtanja.github.io/lab-book/assets/images/githuboctocat.svg)
</code></pre>
<p class="notice--success">We have a winner!</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/githuboctocat.svg" alt="img-png" /></p>
<p>Image that fills page content container by adding the <code class="language-plaintext highlighter-rouge">.full</code> class with:</p>
<p><strong>HTML:</strong></p>
<div class="language-html highlighter-rouge"><div class="highlight"><pre class="highlight"><code><span class="nt"><img</span> <span class="na">src=</span><span class="s">"{{ site.url }}{{ site.baseurl }}/assets/images/githuboctocat.svg"</span> <span class="na">alt=</span><span class="s">""</span> <span class="na">class=</span><span class="s">"full"</span><span class="nt">></span>
</code></pre></div></div>
<p><strong>Kramdown with %raw%:</strong></p>
<div class="language-markdown highlighter-rouge"><div class="highlight"><pre class="highlight"><code><span class="p">![</span><span class="nv">alt</span><span class="p">](</span><span class="sx">{{</span> site.url }}{{ site.baseurl }}/assets/images/githuboctocat.svg)
{: .full}
</code></pre></div></div>
<p class="full"><img src="{{ site.url }}{{ site.baseurl }}/assets/images/githuboctocat.svg" alt="alt" /></p>
<p>isn’t showing in the code fence?</p>
<p><strong>Kramdown:</strong></p>
<div class="language-plaintext highlighter-rouge"><div class="highlight"><pre class="highlight"><code>![img-jpg](https://sarahtanja.github.io/lab-book/assets/images/githuboctocat.svg)
</code></pre></div></div>
<p class="notice--success">We have another winner!</p>
<p class="full"><img src="https://sarahtanja.github.io/lab-book/assets/images/githuboctocat.svg" alt="img-jpg" /></p>
<hr />
<p>Supposedly, you can <a href="https://www.fabriziomusacchio.com/blog/2021-08-11-Minimal_Mistakes_Cheat_Sheet/#via-markdown">embed images</a> in three ways… here I’m just trying to get markdown or html to work (not messing with liquid galleries yet)</p>
<h1 id="via-markdown">Via Markdown</h1>
<p>absolute path - doesn’t render in Typora or Jekyll page, does show in <a href="https://github.com/sarahtanja/lab-book/blob/master/_posts/2023-02-02-test.md">GitHub repo preview</a></p>
<div class="language-plaintext highlighter-rouge"><div class="highlight"><pre class="highlight"><code>![img-svg](https://github.com/sarahtanja/lab-book/blob/master/assets/images/githuboctocat.svg)
</code></pre></div></div>
<p><img src="https://github.com/sarahtanja/lab-book/blob/master/assets/images/githuboctocat.svg" alt="img-svg" /></p>
<p>relative path - this shows in Typora, but nowhere else</p>
<div class="language-plaintext highlighter-rouge"><div class="highlight"><pre class="highlight"><code>![img-png](..\assets\images\githuboctocat.png)
</code></pre></div></div>
<p><img src="..\assets\images\githuboctocat.png" alt="img-png" /></p>
<h1 id="via-html">Via HTML</h1>
<div class="language-plaintext highlighter-rouge"><div class="highlight"><pre class="highlight"><code><figure style="width: 80px" class="align-center">
<a href="/assets/images/githuboctocat.svg" title="octocat" alt="octocat">
<img src="/assets/images/githuboctocat.svg" alt=""></a>
<figcaption>octocat</figcaption>
</figure>
</code></pre></div></div>
<figure style="width: 80px" class="align-center">
<a href="/assets/images/githuboctocat.svg" title="octocat" alt="octocat">
<img src="/assets/images/githuboctocat.svg" alt="" /></a>
<figcaption>octocat</figcaption>
</figure>
<p>This doesn’t show locally, on GitHub preview, or on Jekyll site</p>Sarah S.D. TanjaThis post is for troubleshooting why my images are not rendering on the Minimal Mistakes site.Master’s Thesis Committee Meeting #12023-01-31T00:00:00+00:002023-01-31T00:00:00+00:00https://sarahtanja.github.io/lab-book/committee%20meetings/committee-no1<h2 id="monday-feb-6th-2023"><em>Monday Feb 6th 2023</em></h2>
<h1 id="agenda">Agenda</h1>
<p>Introduction - 5 mins</p>
<ol>
<li>Discuss Committee Agreements - 10 mins</li>
<li>Research Overview - 15 mins</li>
<li>Thesis Direction - 10 mins</li>
<li>Feedback - 20 mins</li>
</ol>
<hr />
<h2 id="1-committee-agreements-first-draft">1. Committee Agreements <em>first draft</em></h2>
<p>Supervisory Committee Chair - Dr. Jacqueline L Padilla-Gamiño, jpgamino@uw.edu</p>
<p>Committee UW SAFS Core Faculty - Dr. Steven B Roberts, sr320@uw.edu</p>
<p>Committee Member - Dr. Jesse R Zaneveld, zaneveld@uw.edu</p>
<p>Graduate Student, (Master’s Thesis) - Sarah S Tanja, stanja@uw.edu</p>
<hr />
<p class="notice--info"><strong>*Note:</strong> These agreements will help facilitate our mentor/mentee expectations in a living document that will be collaboratively updated throughout Sarah’s Master’s work *</p>
<p>From the <a href="https://docs.google.com/document/d/1KITnRsUObcn0QNrKD8WAiq3dozwWW2Bs_fJYRq5u_cQ/edit">2022-23 SAFS graduate student guide:</a></p>
<blockquote>
<p>“The Chair and at least one-half of the total membership must be members of the Graduate Faculty. A minimum of two committee members must also be SAFS Core faculty<strong>.</strong> <strong>The supervisory committee will provide expertise and depth in areas related to the student’s research. The student should rely primarily on the Supervisory Committee Chair and secondarily on the other committee members for professional guidance.</strong>”</p>
</blockquote>
<h3 id="our-menteementor-expectations-are">Our Mentee/Mentor Expectations are:</h3>
<ul>
<li>
<p>We will work with open, honest, and kind communication</p>
</li>
<li>
<p>We will prioritize doing fewer projects well over many small projects poorly</p>
</li>
<li>
<p>We acknowledge that we may be working flexibly or remotely from different time-zones</p>
</li>
<li>
<p>Sarah will organize her research with <code class="language-plaintext highlighter-rouge">GitHub</code> to foster open science</p>
<ul>
<li>tips on <a href="http://paulklemm.com/blog/2014-07-16-use-github-for-scientific-writing/">writing & editing a manuscript</a> with multiple reviewers using<code class="language-plaintext highlighter-rouge">GitHub</code></li>
</ul>
</li>
<li>
<p>Sarah will organize all committee meetings, and be responsible for reaching out to individual committee members for help when she needs it</p>
</li>
<li>
<p>Sarah is on a learning curve and will need to reach out to the committee for guidance; at the same time it is her responsibility to own the project and learn the analyses she is conducting</p>
</li>
<li>
<p>Sarah will maintain an <a href="https://sarahtanja.github.io/lab-book/">open lab notebook</a> and post (at least weekly) to it</p>
</li>
<li>
<p>The committee will meet quarterly</p>
</li>
</ul>
<h3 id="our-shared-vision-of-success-looks-like">Our shared vision of success looks like:</h3>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />two published manuscripts (one for each chapter)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />one published note (the PAE cleavage pilot study)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />two complete thesis chapters</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />graduation by SPRING ‘24</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />reproducible, open-science</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />prioritize researcher safety & environmental health</li>
</ul>
<h3 id="sarahs-progress-toward-a-ms-will-look-like">Sarah’s progress toward a M.S. will look like:</h3>
<table>
<thead>
<tr>
<th>Milestone</th>
<th>Expected</th>
<th>Completed</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="https://docs.google.com/forms/d/e/1FAIpQLSdMCg96RoRiT4yEIAMorzOPT6GqDYFRQcPp0Ttr0D22idJqYg/viewform">Form Committee</a></td>
<td>[AU22] 16DEC22</td>
<td>[WI23] 05JAN23</td>
</tr>
<tr>
<td><a href="https://forms.gle/15siYRGdHKr5dX2B7">Submit Plan of Study</a></td>
<td>[AU22] 16DEC22</td>
<td>[WI23] 05JAN23</td>
</tr>
<tr>
<td><a href="https://docs.google.com/forms/d/e/1FAIpQLScL5Tp4GOxGUTxV2c1-kXW0orPTZEfuFeGtMF4eoJ9LHC9mcw/viewform">Submit Thesis Proposal</a></td>
<td>[AU22] 16DEC22</td>
<td>[WI23]</td>
</tr>
<tr>
<td>End-of-Program Meeting with GPA</td>
<td>[AU23] 01NOV23</td>
<td> </td>
</tr>
<tr>
<td>Submit Thesis Draft</td>
<td>[WI24] 31JAN24</td>
<td> </td>
</tr>
<tr>
<td>Request Final Exam</td>
<td>[WI24] 31JAN24</td>
<td> </td>
</tr>
<tr>
<td>Request Master’s Degree</td>
<td>[WI24] 31JAN24</td>
<td> </td>
</tr>
<tr>
<td>Final Exam: Thesis Seminar + Oral Exam</td>
<td>[SP24] 05APR24</td>
<td> </td>
</tr>
<tr>
<td>Submit Master’s Thesis</td>
<td>[SP24] 31MAY24</td>
<td> </td>
</tr>
<tr>
<td>Commencement</td>
<td>08JUN24</td>
<td> </td>
</tr>
</tbody>
</table>
<div class="notice--success">
<p><strong>Completed Coursework</strong></p>
<table>
<thead>
<tr>
<th style="text-align: left">Qtr</th>
<th>Course</th>
<th>Credits</th>
<th>Grade</th>
</tr>
</thead>
<tbody>
<tr>
<td style="text-align: left">WI21</td>
<td>OCEAN 518-Scientific Writing</td>
<td>2.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">SU21</td>
<td>FHL 446-Marine Botany</td>
<td>9.0</td>
<td>4.0</td>
</tr>
<tr>
<td style="text-align: left">AU21</td>
<td>FISH 511-Topics: Evol&Ecol Plastics Seminar</td>
<td>2.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">AU21</td>
<td>FISH 522-Hot Topics in AFS</td>
<td>2.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">AU21</td>
<td>FISH 552-Intro R Programming</td>
<td>2.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">AU21</td>
<td>FISH 553-ADV R Programming</td>
<td>2.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">AU21</td>
<td>QSCI 482-Statistical Inference in Ecol</td>
<td>5.0</td>
<td>3.8</td>
</tr>
<tr>
<td style="text-align: left">WI22</td>
<td>FISH 521-Research Proposal Writing</td>
<td>4.0</td>
<td>3.7</td>
</tr>
<tr>
<td style="text-align: left">WI22</td>
<td>FISH 700-Masters Thesis</td>
<td>3.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">WI22</td>
<td>OCEAN 531-Marine Phyto Biogeochem</td>
<td>3.0</td>
<td>3.9</td>
</tr>
<tr>
<td style="text-align: left">WI22</td>
<td>FISH 700-Masters Thesis</td>
<td>5.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">WI22</td>
<td>FISH 541-Environmental Physiology</td>
<td>5.0</td>
<td>4.0</td>
</tr>
<tr>
<td style="text-align: left">SU22</td>
<td>FISH 700-Masters Thesis</td>
<td>10.0</td>
<td>CR</td>
</tr>
<tr>
<td style="text-align: left">AU22</td>
<td>FISH 561-Resource Economics & Policy</td>
<td>4.0</td>
<td>4.0</td>
</tr>
<tr>
<td style="text-align: left">AU22</td>
<td>SMEA 500-Human Dimensions of Global Change</td>
<td>3.0</td>
<td>4.0</td>
</tr>
<tr>
<td style="text-align: left">AU22</td>
<td>SMEA 521-Climate Change Governance</td>
<td>3.0</td>
<td>3.7</td>
</tr>
</tbody>
</table>
</div>
<div class="notice">
<p><strong>Audited Coursework</strong></p>
<table>
<thead>
<tr>
<th style="text-align: left">Qtr</th>
<th>Course</th>
</tr>
</thead>
<tbody>
<tr>
<td style="text-align: left">WI22</td>
<td>B BIO 495 C-Investigative Biology</td>
</tr>
<tr>
<td style="text-align: left">WI22</td>
<td>FISH 427 A-Tropical Marine Biology</td>
</tr>
</tbody>
</table>
</div>
<div class="notice--warning">
<p><strong>In-Progress Coursework</strong></p>
<table>
<thead>
<tr>
<th style="text-align: left">Qtr</th>
<th>Course</th>
<th>Credits</th>
</tr>
</thead>
<tbody>
<tr>
<td style="text-align: left">WI23</td>
<td>FISH 539-US Fishery Management & Policy</td>
<td>3.0</td>
</tr>
<tr>
<td style="text-align: left">WI23</td>
<td>FISH 700-Masters Thesis</td>
<td>7.0</td>
</tr>
</tbody>
</table>
</div>
<div class="notice--danger">
<p><strong>To-do Coursework</strong></p>
<table>
<thead>
<tr>
<th style="text-align: left">Qtr</th>
<th>Course</th>
<th>Credits</th>
</tr>
</thead>
<tbody>
<tr>
<td style="text-align: left">SP23</td>
<td>FISH 546-Bioinformatics for Environmental Sciences</td>
<td>3.0</td>
</tr>
<tr>
<td style="text-align: left">WI24</td>
<td>‘5teen’: FISH 578 Bevan Series</td>
<td>2.0</td>
</tr>
</tbody>
</table>
</div>
<h2 id="2-research-overview">2. Research Overview</h2>
<h3 id="motivation">Motivation</h3>
<p>Broadly, my interest in research is motivated by a desire for a healthy world. This is embodied by the ‘<a href="https://www.cdc.gov/onehealth/index.html">one health</a>’ approach, which recognizes the connections between the health of people, animals, and the environment.</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/one-health.jpg" alt="one-health" /><em>infographic sourced from <a href="https://www.frontiersin.org/articles/10.3389/fmars.2022.980705/full">Morrison et al. 2022</a></em></p>
<p class="notice--info"><em><strong>Note:</strong> More about Sarah’s background, research interests & motivations can be found <a href="https://sarahtanja.github.io/lab-book/about/">here</a>.</em></p>
<h3 id="research-topic-background">Research Topic Background</h3>
<p><strong>The goal of my Master’s thesis research is to gain insight into multiple-stressor interactions of warming oceans and plastic pollution on reef-building corals.</strong></p>
<p>Under a CMIP6 climate scenario, it is forecasted that more than 75% of coral reefs will experience <em>yearly severe bleaching</em> before 2070 due to thermal stress. However, hope for resilient corals exists. In a prolonged marine heatwave event on Kiritimati Atoll, the largest coral atoll in the world, 100% of the corals near chronic anthropogenic disturbances died, but in areas that were not already stressed, 90% of corals died. The 10% that survived after bleaching are hypothesized to have recovered due to lower baseline levels of stress and better water quality compared to corals near chronic anthropogenic stressors like pollution and overfishing (Claar et al. 2020).</p>
<p class="notice--info"><strong>Corals are threatened by global stressors, strategies for reducing local stressors may give corals an edge on survival</strong></p>
<p>While coral coverage has declined since the 50s, petroleum-derived plastic production and subsequent environmental plastic pollution have rapidly increased. An estimated 8 million tons of plastic and their associated additives end up in the oceans every year, with unknown impacts to important coastal coral reef ecosystems. Plastics are emerging environmental pollutants of growing concern for coral reefs, and are the source of a suite of environmental contaminants and persistent organic pollutants posing unknown risks to human and ecosystem health (Rochman et al. 2019). Plastics are non-biodegradable, persisting in the environment for centuries. UV radiation and mechanical weathering cause them to become brittle and fragment into microplastics <5mm, and nanoplastics <1mm. As plastics weather mechanically, they also weather chemically by leaching additives not covalently bonded to the plastic polymer resins (Hahladakis et al. 2018).</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/micro-wx.png" alt="weathering-microplastics" /><em>Figure from <a href="https://www.science.org/doi/epdf/10.1126/science.abg5433">Macleod et al. 2021</a></em></p>
<p>The most common plastic additives are a group of chemicals called plasticizers, also known as phthalates or phthalate acid esters (PAE). They are commonly added to PVC to increase flexibility and durability. Phthalates are known to mimic or interfere with hormones that control developmental, reproductive, and immune response in both humans and animals (Maqbool et al. 2016). Despite the presence of phthalates in 95% of tested coral samples (Montano et al. 2020), little is known about how phthalates interfere with coral physiology and reproduction. Previous research shows that plastic leachates experimentally added to plants, bacteria, and algae impair growth, reduce photosynthesis, and shift microbial communities in lab studies (Capolupo et al. 2020; O’Brien et al. 2022; Tetu et al. 2019).</p>
<p>** Plastic pollution, and associated PAE pollution,</p>
<p>Marine heatwaves, plastic manufacturing, and coastal ocean plastic pollution are all predicted to increase (MacLeod et al. 2021; Oliver et al. 2021).</p>
<p>As discussed in Claar et al. (2020), the success of coral recovery after bleaching will depend on levels of chronic anthropogenic stressors like pollution.</p>
<p class="notice--info">I propose to conduct a series of studies that expose coral gametes and coral colonies to the multiple stressors of phthalate pollution and ocean warming, and subsequently measure the physiological responses, holobiont community structure, and gene expression between treatments.</p>
<p>This proposed research aligns with the mission of the School of Aquatic and Fisheries Sciences at the University of Washington, which is to conduct multidisciplinary research and education in order to understand, conserve, and manage marine ecosystems.</p>
<blockquote>
<p><em>REFERENCES</em></p>
<p>Capolupo, Marco, Lisbet Sørensen, Kongalage Don Ranil Jayasena, Andy M. Booth, and Elena
Fabbri. 2020. “Chemical Composition and Ecotoxicity of Plastic and Car Tire Rubber
Leachates to Aquatic Organisms.” Water Research 169 (February): 115270.
https://doi.org/10.1016/j.watres.2019.115270.</p>
<p>Claar, Danielle C., Samuel Starko, Kristina L. Tietjen, Hannah E. Epstein, Ross Cunning, Kim
M. Cobb, Andrew C. Baker, Ruth D. Gates, and Julia K. Baum. 2020. “Dynamic Symbioses
Reveal Pathways to Coral Survival Through Prolonged Heatwaves.” Nature Communications
11 (1): 6097. https://doi.org/10.1038/s41467-020-19169-y.</p>
<p>Hahladakis, John N., Costas A. Velis, Roland Weber, Eleni Iacovidou, and Phil Purnell. 2018.
“An Overview of Chemical Additives Present in Plastics: Migration, Release, Fate and
Environmental Impact During Their Use, Disposal and Recycling.” Journal of Hazardous
Materials 344 (February): 179–99. https://doi.org/10.1016/j.jhazmat.2017.10.014.</p>
<p>MacLeod, Matthew, Hans Peter H. Arp, Mine B. Tekman, and Annika Jahnke. 2021. “The
Global Threat from Plastic Pollution.” Science 373 (6550): 61–65. https://doi.org/10.1126/science.abg5433.</p>
<p>Maqbool, Faheem, Sara Mostafalou, Haji Bahadar, and Mohammad Abdollahi. 2016.
“Review of Endocrine Disorders Associated with Environmental Toxicants and Possible
Involved Mechanisms.” Life Sciences 145 (January): 265–73. https://doi.org/10.1016/j.lfs.2015.10.022.</p>
<p>Marangoni, Laura FB, Eric Beraud, and Christine Ferrier-Pagès. 2021. “Polystyrene
Nanoplastics Impair the Photosynthetic Capacities of Symbiodiniaceae and Promote Coral
Bleaching.” Science of The Total Environment, 152136.</p>
<p>Montano, Simone, Davide Seveso, Davide Maggioni, Paolo Galli, Stefano Corsarini, and
Francesco Saliu. 2020. “Spatial Variability of Phthalates Contamination in the Reef-Building
Corals Porites Lutea, Pocillopora Verrucosa and Pavona Varians.” Marine Pollution Bulletin
155 (June): 111117. https://doi.org/10.1016/j.marpolbul.2020.111117.</p>
<p>O’Brien, Anna M., Tiago F. Lins, Yamin Yang, Megan E. Frederickson, David Sinton, and
Chelsea M. Rochman. 2022. “Microplastics Shift Impacts of Climate Change on a Plant-
Microbe Mutualism: Temperature, CO2, and Tire Wear Particles.” Environmental Research
203 (January): 111727. https://doi.org/10.1016/j.envres.2021.111727.</p>
<p>Oliver, Eric C. J., Jessica A. Benthuysen, Sofia Darmaraki, Markus G. Donat, Alistair J. Hobday,
Neil J. Holbrook, Robert W. Schlegel, and Alex Sen Gupta. 2021. “Marine Heatwaves.” Annual
Review of Marine Science 13 (1): 313–42. <a href="https://doi.org/10.1146/annurev-marine-032720-095144">https://doi.org/10.1146/annurev-marine-032720-095144</a>.</p>
<p>Rochman, Chelsea M., Cole Brookson, Jacqueline Bikker, Natasha Djuric, Arielle Earn,
Kennedy Bucci, Samantha Athey, et al. 2019. “Rethinking Microplastics as a Diverse
Contaminant Suite.” Environmental Toxicology and Chemistry 38 (4): 703–11.
https://doi.org/10.1002/etc.4371.</p>
<p>Tetu, Sasha G., Indrani Sarker, Verena Schrameyer, Russell Pickford, Liam D. H. Elbourne,
Lisa R. Moore, and Ian T. Paulsen. 2019. “Plastic Leachates Impair Growth and Oxygen
Production in Prochlorococcus, the Ocean’s Most Abundant Photosynthetic Bacteria.”
Communications Biology 2 (May): 184. https://doi.org/10.1038/s42003-019-0410-x.</p>
</blockquote>
<h3 id="main-questions">Main Questions</h3>
<ol>
<li>
<p><strong>Does phthalate (PAE) pollution affect <em>M. capitata</em> coral development?</strong></p>
</li>
<li>
<p><strong>What are the single and combined effects of temperature and PAE on <em>M. capitata</em> coral physiological response (transcriptome)?</strong></p>
</li>
<li>
<p><strong>What are the single and combined effects of temperature and PAE on the <em>M. capitata</em> coral microbiome?</strong></p>
</li>
</ol>
<h3 id="pilot-experiments">‘Pilot’ Experiments</h3>
<h4 id="pae-devo--phthalates-stunt-development-in-m-capitata-coral-embryos">PAE-DEVO | Phthalates stunt development in <em>M. capitata</em> coral embryos</h4>
<p>I exposed <em>M. capitata</em> egg-sperm bundles to increasing concentrations of phthalates to assess toxic effects on the development of coral embryos.</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/bundle-vial.png" alt="bundle-bundle-cross" /><em>add caption</em></p>
<p>I used the <a href="https://www.sigmaaldrich.com/US/en/product/supelco/48223?gclid=CjwKCAjw-8qVBhANEiwAfjXLrp8fD-G8yiB_NyrA3OLy3r2jdylgBmHVPUfGFnMp4CMfuVwePGi1dxoCBQIQAvD_BwE">EPA 506 phthalate esters mix 1</a>, which is a 1mL vial that includes the <strong>6 most common phthalates added to consumer products</strong> each at a concentration of 500$\mu$g / mL, <em>(500,000$\mu$g/L)</em> .</p>
<blockquote>
<p>The mix contained the following 6 phthalates:</p>
<blockquote>
<ol>
<li>Benzyl butyl phthalate <strong>(BBP)</strong>
<ul>
<li>detected in 92.7% of 165 water samples</li>
</ul>
</li>
<li>Bis(2-ethylhexyl) adipate <strong>(DEHA)</strong></li>
<li>Bis(2-ethylhexyl) phthalate **(DEHP) **
<ul>
<li>listed on EPA’s Toxics Release Inventory (TRI)</li>
<li>regulated under the Safe Drinking Water Act. The highest concentration allowed, the maximum contaminant level (MCL), is 0.006 mg/L , <strong>(6$\mu$g/L)</strong> in ‘safe’ drinking water</li>
</ul>
</li>
<li>Dibutyl pthalate <strong>(DBP)</strong>
<ul>
<li>listed on EPA’s Toxics Release Inventory (TRI)</li>
</ul>
</li>
<li>Diethyl phthalate <strong>(DEP)</strong></li>
<li>Dimethyl phthalate <strong>(DMP)</strong></li>
</ol>
</blockquote>
</blockquote>
<p>Concentrations of phthalates in environmental sea water samples reported in the literature:</p>
<table>
<thead>
<tr>
<th>Study Area</th>
<th>BBP</th>
<th>DEHA</th>
<th>DEHP</th>
<th>DBP</th>
<th>DEP</th>
<th>DMP</th>
<th>$\sum$PAE</th>
<th>Reference</th>
</tr>
</thead>
<tbody>
<tr>
<td>Review</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>0.5 - 10 $\mu$g/L</td>
<td>Lynch et al 2022</td>
</tr>
<tr>
<td>Tunisia</td>
<td> </td>
<td> </td>
<td><LOD-168$\mu$g/L</td>
<td><LOD-30.5$\mu$g/L</td>
<td><LOD-17.0$\mu$g/L</td>
<td> </td>
<td> </td>
<td>Jebara et al 2021</td>
</tr>
<tr>
<td>Mediterranean Coastal Spain</td>
<td> </td>
<td>0.0021-0.304$\mu$g/L</td>
<td>0.031-0.617$\mu$g/L</td>
<td> </td>
<td>0.024-0.483$\mu$g/L</td>
<td>0.0028 - 0.142 $\mu$g/L</td>
<td> </td>
<td>Sanchez-Avila et al 2012</td>
</tr>
</tbody>
</table>
<blockquote>
<p><em>REFERENCES</em></p>
<p>Lynch, Jennifer M., Katrina Knauer, and Katherine R. Shaw. 2022. “Plastic Additives in the Ocean.” In <em>Plastics and the Ocean</em>, edited by Anthony L. Andrady, 1st ed., 43–76. Wiley. <a href="https://doi.org/10.1002/9781119768432.ch2">https://doi.org/10.1002/9781119768432.ch2</a>.</p>
<p>Jebara, Amel, Ambrogina Albergamo, Rossana Rando, Angela Giorgia Potortì, Vincenzo Lo Turco, Hedi Ben Mansour, and Giuseppa Di Bella. 2021. “Phthalates and Non-Phthalate Plasticizers in Tunisian Marine Samples: Occurrence, Spatial Distribution and Seasonal Variation.” <em>Marine Pollution Bulletin</em> 163 (February): 111967. https://doi.org/10.1016/j.marpolbul.2021.111967.</p>
<p>Sánchez-Avila, Juan, Romà Tauler, and Silvia Lacorte. 2012. “Organic Micropollutants in Coastal Waters from NW Mediterranean Sea: Sources Distribution and Potential Risk.” <em>Environment International</em> 46 (October): 50–62. https://doi.org/10.1016/j.envint.2012.04.013.</p>
</blockquote>
<p>Based on these environmental measurements, I chose to dose the coral gametes at the following concentrations:</p>
<ul>
<li>0$\mu$g/L : control</li>
<li>0.5$\mu$g/L (0.0005$\mu$g/mL): LOD</li>
<li>5$\mu$g/L (0.005$\mu$g/mL) : would be considered acceptable drinking water</li>
<li>50$\mu$g/L (0.05$\mu$g/mL) : common environmentally relevant value</li>
<li>150$\mu$g/L (0.150$\mu$g/mL): max environmental relevance (Jebara et al. 2021, in Tunisia @ 168$\mu$g/L)</li>
</ul>
<p>After dropping bundle-bundle crosses in each prepped 20mL vial treatment, I allowed the bundles to break apart, releasing sperm, and undergo fertilization.</p>
<p>At 4 hours post fertilization, I photographed all the eggs, zygotes, or embryos in each vial using a dissecting scope to determine what proportion of the bundle-bundle cross successfully made it to initial cleavage.</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/cleave-eggs.png" alt="eggs-initial-cleavage" />*M. capitata eggs at 4hpf (hours post fertilization), images taken using an Amscope camera attached to a dissecting scope *</p>
<p>Inital results from an n=3 show the hypothesized ‘non-monotonic’ response pattern</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/a-little.png" alt="graph-results" /><em>Graph showing initial results from PAE-DEVO pilot study. Of interest is the non-monotonic response (very low PAE concentrations significantly reduced proportion of eggs making it to initial cleavage, and were not significantly different compared to very high concentrations of PAE)</em></p>
<p class="notice--success"><strong>In Summary:</strong> the PAE-DEVO experiment resulted in a total of 15 samples, with an n=3, and showed significant differences between the control and all treatment groups. This pilot study may be a candidate for publication as a note in a low-impact factor journal like</p>
<p class="notice--warning"><strong>Limitations:</strong> Sample size is low and study replication is difficult because it relies on access to intact <em>M. capitata</em> egg-sperm bundles, and the use of <a href="https://www.sigmaaldrich.com/US/en/product/supelco/48223?gclid=CjwKCAjw-8qVBhANEiwAfjXLrp8fD-G8yiB_NyrA3OLy3r2jdylgBmHVPUfGFnMp4CMfuVwePGi1dxoCBQIQAvD_BwE">EPA 506 phthalate esters mix 1</a>, which may pose harm to researcher health. If I were to repeat this experiment I would want to make my own leachate out of a common plastic consumer product, similar to the methods used by Tetu et al. 2019. Though this would be safer, it would not provide insight into the actual concentration of PAEs in the treatment.</p>
<p class="notice--info"><em><strong>Note:</strong> I presented this pilot study in a 5-min lighting talk during the AU’22 SAFS Graduate Student Symposium</em></p>
<h4 id="pae-temp--m-capitata-acute-phthalate--thermal-stress-exposure">PAE-TEMP | <em>M. capitata</em> Acute Phthalate & Thermal Stress Exposure</h4>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/graphical-methods.png" alt="pae-temp-graphical-methods" /><em>graphical methods, JPG suggested simplifying diagram</em></p>
<p class="notice--success"><strong>In Summary:</strong> the PAE-TEMP experiment resulted in a total of 130 samples, with an n=10, that were flash-frozen in liquid nitrogen and can be used for DNA/RNA studies.</p>
<p class="notice--warning"><strong>Limitations:</strong> In order to ensure the phthalate concentration was controlled in each treatment, the 20mL scintillation vials were a closed, ‘mesocosm’, and the corals did not receive water changes during the short 48hr exposure. Because of this, I expect we will see degradation between the environmental and experimental control samples. This study can’t account for the fact that corals in Kaneohe Bay may already be exposed to chronic low-levels of PAE pollution, so we can’t say our control baseline is ‘zero PAE exposure’. Replication may be difficult because it relies on the use of <a href="https://www.sigmaaldrich.com/US/en/product/supelco/48223?gclid=CjwKCAjw-8qVBhANEiwAfjXLrp8fD-G8yiB_NyrA3OLy3r2jdylgBmHVPUfGFnMp4CMfuVwePGi1dxoCBQIQAvD_BwE">EPA 506 phthalate esters mix 1</a>, which may pose harm to researcher health.</p>
<h2 id="3-thesis-direction">3. Thesis Direction</h2>
<p>Of the two pilot experiments, the <em>M. capitata</em> Acute Phthalate & Thermal Stress Exposure (PAE-TEMP) provides the most samples and potential to move toward publishable manuscripts.</p>
<h3 id="chapter-1---microbiome">Chapter 1 - Microbiome</h3>
<div class="notice--info">
<p><strong>Acute phthalate and thermal stress exposure effects on <em>M. capitata</em> coral microbiome</strong></p>
<ul>
<li>submit as a manuscript to <a href="https://www.sciencedirect.com/journal/environmental-pollution">Environmental Pollution</a> (IF 9.988)</li>
<li>author list (Tanja, SS; Roberts, SB; Zaneveld, JR; Padilla-Gamiño, JL)</li>
</ul>
</div>
<div class="notice--warning">
<p><strong>Hypothesis:</strong> Are there any microbes that may be more sensible to temp, PAE or the combination of both?</p>
<ul>
<li>“elevated temperatures make corals more vulnerable to opportunistic bacteria”-<a href="https://www.nature.com/articles/ncomms11833">Zaneveld et al. 2016</a></li>
<li>“Overall, coral microbiome diversity and composition varied with host species, local disturbance and heat stress, but not always as predicted.”-<a href="https://link-springer-com.offcampus.lib.washington.edu/article/10.1007/s00338-019-01779-8#Sec7">McDevitt-Irwin et al 2019</a></li>
<li>“Both heat stress exposures induced the significant structural reorganization of coral-associated bacteria, with bacterial diversity and community heterogeneity significantly increasing with the temperature treatment. ”- <a href="https://www.sciencedirect.com/science/article/abs/pii/S0141113623000284">Zhu et al. 2023</a></li>
</ul>
</div>
<p><strong>Methods:</strong></p>
<ul>
<li><a href="https://www.zymoresearch.com/products/quick-dna-rna-miniprep-plus-kit">Quick DNA/RNA Miniprep Plus Kit</a>
<ul>
<li>50 preps $438.90 , 10 preps $106.70</li>
</ul>
</li>
<li>16S rRNA amplicon sequencing <em>send subsample of control vs. overdose to look for initial differences</em>
<ul>
<li>UW Northwest Genomics Center $__/sample</li>
<li>Azenta</li>
<li>Univ. of Texas</li>
<li><a href="https://www.mrdnalab.com/16s-ribosomal-sequencing.html">Mr. DNA Lab</a> $90/sample for 5k sequences per assay</li>
</ul>
</li>
<li>QIIME pipeline</li>
</ul>
<h3 id="chapter-2---transcriptome">Chapter 2 - Transcriptome</h3>
<div class="notice--info">
<p><strong>Acute phthalate and thermal stress exposure effects on <em>M. capitata</em> coral transcriptome</strong></p>
<ul>
<li>submit as a manuscript to <a href="https://www.sciencedirect.com/journal/environmental-pollution">Environmental Pollution</a> (IF 9.988)</li>
<li>author list (Tanja, SS; Roberts, SB; Zaneveld, JR; Padilla-Gamiño, JL)</li>
</ul>
</div>
<div class="notice--warning">
<p><strong>Hypothesis:</strong> “What genes do you expect to be upregulated or downregulated by temp, PAE, and temp and PAE? and why?”</p>
<ul>
<li><a href="https://onlinelibrary.wiley.com/doi/10.1002/9781118828502.ch9">“Endocrine-like signaling in corals”</a> , book chapter by Ann Tarrant</li>
<li>“components of the transcriptome were significantly upregulated within 90 min and after a temperature increase of +2 degrees C. The developmental transcription factor, Kruppel-like factor 7, was highly expressed within 60 min, and stress-related transcription factors such as Elk-3 were highly expressed starting at 240 min. The sets of genes enriched for early transcriptional response to heat stress included heat shock proteins, small GTPases, and proteasome genes. Retrovirus-related Pol polyproteins from transposons were significantly expressed throughout the whole experiment.” - <a href="https://www-journals-uchicago-edu.offcampus.lib.washington.edu/doi/10.1086/692717">Traylor-Knowles et al. 2017</a></li>
<li>“Overall, redox regulation and metabolite transport are key components of the coral animal thermal stress phenome.” - <a href="https://www-webofscience-com.offcampus.lib.washington.edu/wos/woscc/full-record/WOS:000719047500002">Williams et al. 2021</a></li>
</ul>
</div>
<p><strong>Methods:</strong></p>
<ul>
<li><a href="https://www.zymoresearch.com/products/quick-dna-rna-miniprep-plus-kit">Quick DNA/RNA Miniprep Plus Kit</a>
<ul>
<li>50 preps $438.90 , 10 preps $106.70</li>
</ul>
</li>
<li>RNAseq: <em>send subsample of control vs. overdose to look for initial differences</em>
<ul>
<li>UW Northwest Genomics Center $200/sample <a href="https://github.com/RobertsLab/resources/issues/1543">RobertsLab Get Seq Quote - coral samples issue</a></li>
<li><a href="https://web.azenta.com/promotions">Azenta 50% off promotion</a> $140/sample</li>
<li>Univ. of Texas</li>
<li>Mr. DNA Lab</li>
</ul>
</li>
<li>Linux pipeline?</li>
</ul>
<h3 id="timeline">Timeline</h3>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/gantt-timeline.png" alt="gantt-timeline" /></p>
<h2 id="5-feedback">5. Feedback</h2>
<p>Q. Is the PAE + Temp multiple stressor study rigorous enough to be turned into two manuscripts?
A. potentially, it will depend on what we see in the data. It will be enough for two thesis chapters.</p>
<p>Q. Which sequencing strategy would be best suited to this project?
- Where have you sent samples for sequencing services in the past?
- What are reasonable sequencing costs per sample?</p>
<p>Q. I need support/resources to learn about:
- requesting quotes for sequencing (sequencing platform, # of sequences, # of bp, read-length/assay-length)
- <a href="https://www.idtdna.com/pages/technology/next-generation-sequencing/library-preparation#:~:text=Library%20preparation%20is%20the%20first,and%20tagmentation%2Dbased%20library%20prep.">library preparation</a></p>
<div class="language-plaintext highlighter-rouge"><div class="highlight"><pre class="highlight"><code> > “sequencing libraries are pools of DNA fragments containing adapter sequences compatible with a specific sequencing platform and indexing barcodes for individual sample identification. The main library preparation methods are [ligation-based library preparation](https://www.idtdna.com/pages/technology/next-generation-sequencing/library-preparation/ligation-based-library-prep) tagmentation-based library preparation, and [amplicon library preparation](https://www.idtdna.com/pages/technology/next-generation-sequencing/dna-sequencing/targeted-sequencing/amplicon-sequencing). The specific protocol you choose depends on your sequencing platform and downstream analysis.”
- large file storage (Hyak super computer at SAFS?) *got stuck at this step when working with microbiome data last Winter*
- [RNAseq pipeline](https://www.azenta.com/blog/quick-start-guide-rna-seq-data-analysis)
</code></pre></div></div>
<blockquote>
<p>General comments from committee: just jump in and get going with DNA/RNA extraction, this needs to be done ASAP</p>
</blockquote>Sarah S.D. TanjaMonday Feb 6th 2023CV2023-01-27T00:00:00+00:002023-01-27T00:00:00+00:00https://sarahtanja.github.io/lab-book/blog/cv<h1 id="sarah-sd-tanja">Sarah S.D. Tanja</h1>
<div style="text-align:center"> Graduate Student Researcher | Padilla-Gamiño Lab </div>
<div style="text-align:center">University of Washington | School of Aquatic & Fishery Sciences</div>
<div style="text-align:center">1122 NE Boat Street, Box 355020, Seattle, WA 98195 USA</div>
<div style="text-align:center">e-mail: stanja@uw.edu</div>
<blockquote>
<h4 id="links"><em>links</em></h4>
<p>:notebook: Open Lab Notebook: https://sarahtanja.github.io/lab-book/
<img src="C:\Users\Minerva\Documents\GitHub\lab-book\assets\images\githuboctocat.svg" style="zoom:9%;" /> GitHub: https://github.com/sarahtanja
<img src="C:\Users\Minerva\Documents\GitHub\lab-book\assets\images\coral-icon" style="zoom:8%;" /> Padilla-Gamiño Lab: https://padilla-gaminolab.weebly.com/</p>
</blockquote>
<h4 id="about">ABOUT</h4>
<h4 id="education">EDUCATION</h4>
<p><strong>M. S. in Aquatic & Fishery Sciences, University of Washington; Seattle, WA</strong> (<em>anticipated 2024</em>)</p>
<p><strong>B. S. in Biology with Honors, University of Portland; Portland, OR (2015)</strong></p>
<h4 id="awards--honors">AWARDS & HONORS</h4>
<h4 id="scholarships--grants">SCHOLARSHIPS & GRANTS</h4>
<h4 id="professional-experience">PROFESSIONAL EXPERIENCE</h4>
<h4 id="field-work">FIELD WORK</h4>
<h4 id="science-communication-service--outreach">SCIENCE COMMUNICATION, SERVICE & OUTREACH</h4>
<h4 id="mentorship">MENTORSHIP</h4>
<h4 id="certifications">CERTIFICATIONS</h4>Sarah S.D. TanjaSarah S.D. TanjaAbout ‘Omics2023-01-13T00:00:00+00:002023-01-13T00:00:00+00:00https://sarahtanja.github.io/lab-book/blog/about-omics<h2 id="three-domains-of-life">three domains of life</h2>
<ul>
<li>
<p>Eukaryotes - nucleus</p>
</li>
<li>
<p>Bacteria - no nucleus</p>
</li>
<li>
<p>Archaea - no nucleus</p>
</li>
</ul>
<h2 id="the-central-dogma-of-biology">the central dogma of biology</h2>
<p>DNA —>[<a href="https://www.labxchange.org/library/items/lb:LabXchange:1d4afb7e:lx_simulation:1"><code class="language-plaintext highlighter-rouge">transcription</code></a>]—> mRNA—>[<a href="https://www.labxchange.org/library/items/lb:LabXchange:049914b8:lx_simulation:1"><code class="language-plaintext highlighter-rouge">translation</code></a>]—> polypeptide-chain—>[modification]—>protein</p>
<p>Differences in <a href="https://youtu.be/WNZf4ip_R9s"><code class="language-plaintext highlighter-rouge">translation</code></a> between eukaryotes (human cell) and prokaryotes (bacterium):</p>
<p><img src="https://sarahtanja.github.io/lab-book\assets\images\eu-vs-pro.png" alt="eukaryote-prokaryote-cells" /><em>figure source: <a href="https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/translation/a/intro-to-gene-expression-central-dogma">Khan Academy AP College Biology Unit 6 Lesson 4</a></em></p>
<h2 id="definitions">definitions</h2>
<table>
<thead>
<tr>
<th>jargon</th>
<th>description</th>
</tr>
</thead>
<tbody>
<tr>
<td>bp</td>
<td>Base-pairs (G-C) (A-T)</td>
</tr>
<tr>
<td>epigenomics</td>
<td>Modifications to DNA that affect gene expression without altering the DNA sequence; primarily DNA methylation and histone modification</td>
</tr>
<tr>
<td>genome</td>
<td>An organism’s complete set of DNA sequences across all chromosomes, including all genes, and ultimately the three-dimensional form that it takes</td>
</tr>
<tr>
<td>microbiome</td>
<td>The genomes of bacteria, archaea, and fungi that collectively reside in an environment. The microbiome can be investigated using Shotgun Metagenome Sequencing and 16S rRNA Sequencing.</td>
</tr>
<tr>
<td>microbiota</td>
<td>The living community of archaea, bacteria, and fungi in an environment</td>
</tr>
<tr>
<td>transcriptome</td>
<td>An organism’s complete set of RNA, a collection of all the transcript readouts present in a cell</td>
</tr>
<tr>
<td>Mb / Mbp</td>
<td>Genome size is the total amount of <a href="https://en.wikipedia.org/wiki/DNA">DNA</a> contained within one copy of a single complete <a href="https://en.wikipedia.org/wiki/Genome">genome</a> and can be measured as the total number of <a href="https://en.wikipedia.org/wiki/Nucleotide">nucleotide</a> <a href="https://en.wikipedia.org/wiki/Base_pair">base pairs</a>, usually in megabases (millions of base pairs, abbreviated Mb or Mbp)</td>
</tr>
</tbody>
</table>
<h2 id="16s-rrna-amplicon-sequencing">16s rRNA amplicon sequencing</h2>
<p>WHY Choose 16S over Shotgun Metagenomic Sequencing? WATCH THIS ➡️ <a href="https://www.youtube.com/watch?v=kXYJ_Dc9qcc">https://www.youtube.com/watch?v=kXYJ_Dc9qcc</a></p>
<h2 id="rna-seq">RNA-seq</h2>
<p>WATCH THIS ➡️ <a href="https://www.youtube.com/watch?v=tlf6wYJrwKY">https://www.youtube.com/watch?v=tlf6wYJrwKY</a></p>
<p>READ THIS ➡️ <a href="https://hbctraining.github.io/Intro-to-rnaseq-hpc-salmon/lessons/Intro-to-RNAseq.html">Introduction to RNA-seq using high-performance computing</a>, was made by the Harvard Chan Bioinformatics Core <a href="https://github.com/hbctraining">github.com/hbctraining</a> 👌🙌.</p>
<p>For even more detail, READ THIS lesson material ➡️ <a href="https://scienceparkstudygroup.github.io/rna-seq-lesson/">https://scienceparkstudygroup.github.io/rna-seq-lesson/</a> “Bliek Tijs, Frans van der Kloet and Marc Galland” (eds): “RNA-seq lesson.” Version 2020.04. https://github.com/ScienceParkStudyGroup/rnaseq-lesson</p>
<p>Simply put, RNA-seq (Ribonucleic Acid Sequencing) can quantify gene expression: “which genes are turned on (active) and, <em>how much</em> they are transcribed.”</p>
<p>The transcriptome is defined as a collection of all the transcript readouts present in a cell. RNA-seq data can be used to explore and/or quantify the transcriptome of an organism, which can be utilized for the following types of experiments:</p>
<ul>
<li>
<p>Differential Gene Expression: <em>quantitative</em> evaluation and comparison of transcript levels</p>
</li>
<li>
<p>Transcriptome assembly: building the profile of transcribed regions of the genome, a <em>qualitative</em> evaluation.</p>
</li>
<li>
<p>Can be used to help build better gene models, and verify them using the assembly</p>
</li>
<li>
<p>Metatranscriptomics or community transcriptome analysis</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/gene-structure.png" alt="gene-structure" /></p>
</li>
</ul>
<h3 id="single-nucleotide-polymorphisms-snps">Single Nucleotide Polymorphisms (SNPs)</h3>
<h3 id="read-depth-read-per-sample">Read Depth/ Read per Sample</h3>
<p>The number of times a particular base is represented within all the reads from sequencing. The higher the read depth, the more confidence scientists can have in identifying a base – known as ‘base calling’. By sequencing each fragment numerous times to produce multiple reads, scientists can be more confident that any <a href="https://www.genomicseducation.hee.nhs.uk/glossary/variant/">variants</a> identified are true variants and not <a href="https://www.genomicseducation.hee.nhs.uk/glossary/artefact/">artefacts</a> from the sequencing process. The number of times each individual base has been sequenced i.e. the number of reads it appears in is referred to as the read depth, and the greater the depth, the more confident scientists can be that the variant is real.</p>
<p>*Generally, we recommend 5-10 million read pairs per sample for small genomes (e.g. bacteria) and 20-30 million read pairs per sample for large genomes (e.g. human, mouse). Medium genomes often depend on the project, but 15-20 million read pairs per sample is typically sufficient. For de novo transcriptome assembly projects, we recommend 100 million read pairs per sample.</p>
<p><img src="https://sarahtanja.github.io/lab-book/assets/images/coverage.png" alt="read-depth" />[https://www.metagenomics.wiki/pdf/qc/coverage-read-depth]</p>
<h3 id="total-rna-sample-submission-requirements">Total RNA Sample Submission Requirements</h3>
<table>
<thead>
<tr>
<th>company</th>
<th>min reads per sample</th>
<th> </th>
<th>sample type</th>
<th>min RNA amount</th>
<th>recommended RNA amount</th>
<th>Purity (A260/280)</th>
<th>RIN</th>
<th>buffer</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="https://web.genewiz.com/rna-seq-faq?utm_term=&utm_campaign=NGS+2018&utm_source=adwords&utm_medium=ppc&hsa_tgt=dsa-1481494939850&hsa_grp=73632456640&hsa_src=g&hsa_net=adwords&hsa_mt=&hsa_ver=3&hsa_ad=487231038637&hsa_acc=8363678060&hsa_kw=&hsa_cam=1038345344&gclid=Cj0KCQjwuLShBhC_ARIsAFod4fLsx04Oza0_CdPHEJQpgTP97tEZWU2uy6PBn16cCAJ08oSRGPu9lRQaAmD1EALw_wcB">Azenta</a> (Genewiz)</td>
<td> </td>
<td> </td>
<td>Total RNA</td>
<td>500 ng</td>
<td>>2 ug , >50ng/uL</td>
<td>1.8 - 2.2</td>
<td>>=6.0</td>
<td>nuclease-free water</td>
</tr>
<tr>
<td>UW Northwest Genomics Center</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>University of Texas</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mr. DNA Lab</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>FYR Diagnostics</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
<h4 id="what-method-is-used-to-remove-ribosomal-rna">What method is used to remove ribosomal RNA?</h4>
<p>Since ribosomal RNA (rRNA) makes up the majority of total RNA, its removal is necessary for efficient sequencing of other RNA species, such as mRNA, long non-coding RNA (lncRNA), and small RNA.</p>
<ul>
<li>
<p>For Standard and Strand-Specific RNA-Seq, you can select either poly-A selection or rRNA depletion methods. Poly-A selection is sufficient for studying mRNA in eukaryotes. Analysis of lncRNA or bacterial transcripts requires rRNA depletion.</p>
</li>
<li>
<p>For Ultra-Low Input RNA-Seq, the default is to use poly-A selection. However, if your project requires analysis of lncRNA in addition to mRNA, please make a comment on the quote request form, and we can discuss the available options.</p>
</li>
</ul>
<p>### 1. Illumina library preparation</p>
<p>To do RNA-seq, we have to isolate RNA from each sample and turn it into cDNA (complementary). <strong>The cDNA that we make is called the ‘library’</strong>. Why is a collection of cDNA a library?</p>
<p>The cDNA libraries can be generated in a way to retain information about which strand of DNA the RNA was transcribed from.</p>
<p>Libraries that retain this information are called stranded libraries, which are now standard with Illumina’s TruSeq stranded RNA-Seq kits. Stranded libraries should not be any more expensive than unstranded, so there is not really any reason not to acquire this additional information.</p>
<p>There are 3 types of cDNA libraries available:</p>
<ul>
<li>Forward – reads resemble the gene sequence</li>
<li>Reverse – reads resemble the complement of the gene sequence (TruSeq)</li>
<li>Unstranded</li>
</ul>
<p>To make the cDNA library:</p>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />isolate mRNA or total RNA</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />remove contaminant DNA</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />either remove rRNA( ribosomal RNA depletion) or select mRNA (polyA selection), resulting in fragmented RNA</p>
<blockquote>
<p><strong>For differential gene expression analysis, it is best to enrich for Poly(A)+</strong></p>
</blockquote>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />reverse transcribe the fragmented RNA into double-stranded cDNA</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Add (ligate) sequencing adaptors</p>
<blockquote>
<p><strong>Allows sequencing machine to recognize the fragments and to sequence many samples at the same time, different samples can use different adaptors</strong></p>
</blockquote>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />PCR amplify the library</p>
<blockquote>
<p><strong>Only the fragments with sequencing adaptors are amplified</strong></p>
</blockquote>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />fragments are size selected (usually 300-500bp)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Quality Control</p>
</li>
</ul>
<hr />
<p>### 2. Illumina Sequencing</p>
<p>Fragments are laid out vertically in a grid, called a flow cell. Fluorescent probes</p>
<p>Low quality score</p>
<ul>
<li>
<p>Low confidence : fluorescent probe didn’t shine bright enough</p>
</li>
<li>
<p>Low diversity : overabundance of a single color making it hard to identify individual sequences (the colors blue together) especially problematic in the first few nucleotidesof a sequence because that is when the sequencer determines where the DNA fragments are located on the flow cell</p>
</li>
</ul>
<p>Each sequencing read has 4 lines of data:</p>
<p>1ST LINE: Unique ID for sequence</p>
<p>2ND LINE: Bases for sequenced fragment ex. (ACAGACGGATGACGA)</p>
<p>3RD LINE: ‘+’ spacer character</p>
<p>4TH LINE: quality scores for each base in the sequenced fragments</p>
<p>Filter out garbage reads</p>
<ul>
<li>reads with low quality base calls</li>
<li>reads that are adaptor sequences bound to each other (no DNA fragment)</li>
</ul>
<p>Align high quality reads to a genome</p>
<ul>
<li>Index of all fragments and locations in the genome</li>
</ul>
<p>Count the number of reads per gene</p>
<ul>
<li>‘Bulk’ RNA-seq sample is the average of a pool of the same treatment</li>
</ul>
<p>Normalize the data</p>
<ul>
<li>low quality reads</li>
<li>how many reads were successful</li>
</ul>
<hr />
<h3 id="3-data-analysis">3. Data Analysis</h3>
<p>The data is a huge matrix.</p>
<p><a href="https://www.youtube.com/watch?v=FgakZw6K1QQ">Principal component analysis (PCA)</a></p>
<p><a href="https://marineomics.github.io/FUN_01_DGE_comparison_v2.html">Marine Omics Fitting Multifactorial Models of Differential Expression, RNA-seq</a></p>Sarah S.D. Tanjathree domains of lifeDual DNA & RNA Extraction Protocol2023-01-13T00:00:00+00:002023-01-13T00:00:00+00:00https://sarahtanja.github.io/lab-book/protocols/protocol-dna-rna-extraction<p>The aim of this protocol is to take coral fragments that have been snap-frozen in liquid nitrogen and extract both DNA for downstream archaea & bacteria microbiome 16S Microbiome Sequencing and RNA for downstream coral-host tissue Tag-seq or RNA-seq analysis.</p>
<blockquote>
<p>In plain, I want to answer ‘who is there?’ regarding the bacterial community, and ‘what is the coral doing?’ regarding gene expression.</p>
</blockquote>
<p>The benefits of doing DNA and RNA extraction together are that it saves time, money, & sample material. It also facilitates paired sample analysis, where each data point in one dataset is uniquely paired to a data point in the second dataset because we are making duplicate measurements on the <em>same sample</em>.</p>
<p>The challenge is lysing the sample enough to get the bacterial DNA (busting open all those layers of cell membranes!) and retaining host (coral) RNA integrity.</p>
<p>This protocol relies heavily on the Zymo Research <a href="https://files.zymoresearch.com/protocols/_d7003t_d7003_quick-dna-rna_miniprep_plus_kit.pdf">Protocol PDF</a> & URI Putnam Lab Emma Strand’s notebook post on ‘<a href="https://emmastrand.github.io/EmmaStrand_Notebook/Zymo-Duet-RNA-DNA-Extraction-Protocol/">Zymo-Duet-RNA-DNA-Extraction-Protocol</a>’, with a few alterations suggested by Zymo tech on <a href="https://sarahtanja.github.io/lab-book/blog/daily-log/#28-mar-2023-t">March 28th 2023</a> regarding <a href="https://files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf">optimized lysis protocols</a>.</p>
<p>📞Zymo is very responsive to phone calls! Any questions on the kit you can call <strong>888-882-9682</strong> and reference catalog number <strong>D7003</strong> to ask technical questions galore.</p>
<h2 id="advanced-prep">Advanced Prep</h2>
<h3 id="biological-sample-info">Biological Sample Info</h3>
<ul>
<li>liquid-nitrogen snap-frozen ~3cm <em>Montipora capitata</em> coral fragments held at -80°C</li>
</ul>
<h3 id="background">Background</h3>
<p>👀 WATCH ➡️ <a href="https://www.youtube.com/watch?v=Y_-xP60SYe0">Bumbling Biochemist ‘Spin column purification of nucleic acids’</a> on YouTube</p>
<p>👀 WATCH ➡️ <a href="https://www.youtube.com/watch?v=MBnuae5aWg4">Bumbling Biochemist ‘tips for working with DNA/RNA spin columns’</a> on YouTube</p>
<p>📖 READ ➡️ <a href="https://biolinkk.com/posts/can-coral-dna-measure-ocean-health/">CAN CORAL DNA MEASURE OCEAN HEALTH?</a> on biolinkk</p>
<h3 id="materials-list">Materials List</h3>
<h4 id="extraction-kit"><em>Extraction Kit</em></h4>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Zymo Research <a href="https://www.zymoresearch.com/products/quick-dna-rna-miniprep-plus-kit">Quick-DNA/RNA Miniprep Plus Kit</a> (50 prep D7003, or 10 prep D7003T)<em>1 prep = 1 sample that results in DNA in one tube, and RNA in another</em> <em>includes:</em></p>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DNA/RNA Lysis Buffer</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DNA/RNA Prep Buffer</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DNase/RNase-Free Water</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DNase I2 (lyophilized… aka freeze-dried)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DNA Digestion Buffer</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DNA/RNA Shield (2X concentrate)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />PK Digestion Buffer</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Proteinase K3 (lyophilized… aka freeze-dried)</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Proteinase K3 Storage Buffer</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Spin-Away Filters</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Zymo-Spin IIICG Columns</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Collection Tubes</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.zymoresearch.com/collections/lysis-tubes/products/zr-bashingbead-lysis-tubes-0-1-0-5-mm">ZR BashingBead Lysis 2mL Tubes with 0.1 & 0.5mm beads</a>, 1 per prep</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.zymoresearch.com/products/dnase-rnase-free-tubes">DNase/RNase/nuclease-Free Tubes</a>, 3 per prep</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.zymoresearch.com/products/dnase-rnase-free-tubes">DNase/RNase-Free Tubes</a>, 3 per prep</p>
</li>
</ul>
<h4 id="ppe"><em>PPE</em></h4>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />nitrile gloves</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />liquid-nitrogen & cold storage handling gloves</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />lab coats</li>
</ul>
<h4 id="lab-equipment"><em>Lab Equipment</em></h4>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.amazon.com/dp/B09L3Q99WL/ref=sspa_dk_hqp_detail_aax_0?psc=1&sp_csd=d2lkZ2V0TmFtZT1zcF9ocXBfc2hhcmVk&spLa=ZW5jcnlwdGVkUXVhbGlmaWVyPUEyWUpaVTc1M0pRMVImZW5jcnlwdGVkSWQ9QTAxMTgzODBJOFI3QlZFQ0pKQUsmZW5jcnlwdGVkQWRJZD1BMDc2MTE0M1ExRUdBSkEwOFBBRiZ3aWRnZXROYW1lPXNwX2hxcF9zaGFyZWQmYWN0aW9uPWNsaWNrUmVkaXJlY3QmZG9Ob3RMb2dDbGljaz10cnVl">ethanol-proof lab markers</a></p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" /><a href="https://www.southernlabware.com/80-well-micro-tube-racks-assorted-5-pacl.html?gclid=CjwKCAiAjPyfBhBMEiwAB2CCIkxZXHoOz72MNKvsWFcKscbsG8H5wIyMEw974wfKSvbU2W5a4Tk2IRoCBtEQAvD_BwE">1.5mL microcentrifuge tube racks</a></p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />lab mortar & pestle sets</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />lab label tape</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />centrifuge</p>
</li>
<li class="task-list-item">
<p>[ ]Beckman Coulter Microfuge 16](<a href="https://www.beckman.com/landing/ppc/cent/benchtop/microcentrifuges?utm_source=google&utm_medium=cpc&utm_term=microcentrifuge&gclid=CjwKCAjw586hBhBrEiwAQYEnHYq-F4WNopG5rWYG4Bb0YjPlfXL7pVCESNmTwqyT6Cgxty-Si8AAdhoC8I0QAvD_BwE">https://www.beckman.com/landing/ppc/cent/benchtop/microcentrifuges?utm_source=google&utm_medium=cpc&utm_term=microcentrifuge&gclid=CjwKCAjw586hBhBrEiwAQYEnHYq-F4WNopG5rWYG4Bb0YjPlfXL7pVCESNmTwqyT6Cgxty-Si8AAdhoC8I0QAvD_BwE</a>)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />mortexer / homogenizer</p>
<ul>
<li>
<p><a href="https://www.southernlabware.com/mortexertm-vortex-mixer-115v.html?gclid=CjwKCAjw586hBhBrEiwAQYEnHZGNGUTMUHNWEHBx5r2Ig9BZSuRVSnvE2_OV6n0mGga5fqV8DPcjfRoCsNoQAvD_BwE">Mortexer Vortex Mixer</a> with included Multi-Head to hold 8 microcentrifuge tubes (this is what we have)</p>
<p>…OR…</p>
</li>
<li>
<p>Vortex Genie 2 with <a href="https://www.zymoresearch.com/products/horizontal-microtube-holder">Horizontal Microtube Holder</a> (recommended by <a href="https://files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf">Zymo optimized lysis protocols</a>)</p>
</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />liquid-nitrogen (stored in dewer or tank)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />small liquid-nitrogen thermos (1L)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />small cooler (for dry ice and samples)</p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />##### dry-ice</p>
<ul>
<li>
<p>Biochemistry Store in hallway adjacent to Room J-014, Health Sciences Building</p>
</li>
<li>
<p>The entrance can be found at the <a href="https://goo.gl/maps/hYnYcm6EiTHGEE4SA">Health Sciences Building loading dock</a> across NE Boat St. from Saint Bread, NE of the Ocean Sciences Building</p>
</li>
<li>
<p>must have UW Husky Card to fob-activate doors leading from loading dock and interior hall</p>
</li>
<li>
<p>bring well-insulated foam or hard cooler. First weigh empty cooler, add dry-ice, then weigh again and record final weight, budget#, PI, and name on the clipboard near the dry-ice station</p>
</li>
<li>
<p>probably best to drive rather than carry a cooler of dry-ice for three blocks</p>
</li>
</ul>
</li>
</ul>
<h4 id="reagents"><em>Reagents</em></h4>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />nuclease free (<a href="https://www.thermofisher.com/order/catalog/product/4387937#:~:text=DEPC%2Dtreated%20water%20is%20autoclaved,%2C%20exonuclease%2C%20and%20RNase%20activity.">DEPC-treated</a>) autoclaved water</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />95% (190 proof) - 99.5% (200 proof) ethanol</li>
</ul>
<h4 id="sterilizing"><em>Sterilizing</em></h4>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />10% bleach in spray-bottle</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />70% ethanol in spray-bottle</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />DI water in spray-bottle</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />RNase away in spray-bottle</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Kimwipes/paper towels</li>
</ul>
<h4 id="pipettes--tips"><em>Pipettes & Tips</em></h4>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P10 + filtered tips <em>DNase/RNase free</em></li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P100 + filtered tips <em>DNase/RNase free</em></li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P1000 + filtered tips <em>DNase/RNase free</em></li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />P5000 + tips (for buffer prep)</li>
</ul>
<h3 id="randomize-sample-processing">Randomize Sample Processing</h3>
<p>Make sure to randomize which samples are processed in each batch to reduce ‘batch effects’!</p>
<p>See <a href="https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/rand-sample-processing.Rmd">‘Randomize Sample Processing’</a> script as an example.</p>
<p>Think about how many samples you can process at once, and your kit, centrifuge, and homogenizer capacity.</p>
<h2 id="lab-setup">Lab Setup</h2>
<h3 id="sterilize">Sterilize</h3>
<ol>
<li>
<p>Don lab coat 🥼 & tie hair back , glove up 🧤</p>
</li>
<li>
<p>Spray down <code class="language-plaintext highlighter-rouge">benchtop</code>, <code class="language-plaintext highlighter-rouge">microcentrifuge tube racks</code>, <code class="language-plaintext highlighter-rouge">pipettes</code>, and <code class="language-plaintext highlighter-rouge">pipette tip boxes</code> with:</p>
<ul>
<li>
<p><code class="language-plaintext highlighter-rouge">10% bleach</code> in spray-bottle, then wipe with Kimwipe</p>
</li>
<li>
<p><code class="language-plaintext highlighter-rouge">DI water</code> in spray-bottle, then wipe with Kimwipe</p>
</li>
<li>
<p><code class="language-plaintext highlighter-rouge">70% ethanol</code>in spray-bottle, then wipe with Kimwipe</p>
</li>
</ul>
</li>
<li>
<p>Spray down <code class="language-plaintext highlighter-rouge">mortars & pestles</code> , <code class="language-plaintext highlighter-rouge">scoopulas</code>, and <code class="language-plaintext highlighter-rouge">forceps</code> with:</p>
<ul>
<li>
<p><code class="language-plaintext highlighter-rouge">10% bleach</code> in spray-bottle, then wipe with Kimwipe</p>
</li>
<li>
<p><code class="language-plaintext highlighter-rouge">DI water</code> in spray-bottle, then wipe with Kimwipe</p>
</li>
<li>
<p><code class="language-plaintext highlighter-rouge">70% ethanol</code> in spray-bottle, then wipe with Kimwipe</p>
</li>
<li>
<p><code class="language-plaintext highlighter-rouge">RNase away</code> in spray-bottle, then wipe with Kimwipe</p>
</li>
</ul>
</li>
<li>
<p>Spray Kimwipe with <code class="language-plaintext highlighter-rouge">RNase away</code> and wipe down equipment buttons/handles/surfaces that may have been touched by ungloved hands</p>
</li>
<li>
<p>Spray <code class="language-plaintext highlighter-rouge">RNase away</code>on gloves and rub hands together</p>
</li>
</ol>
<h3 id="arrange-lab-bench">Arrange Lab Bench</h3>
<p>This extraction protocol can be split into two main phases: 1. lysing and 2. purification. I setup the lab bench with each phase occupying a station on the bench, with materials used in each phase arranged accordingly. Make sure you have trash, pipette disposal, and waste disposal containers within easy reach of the workstation.</p>
<p>Lysing Station</p>
<ul>
<li>
<p>liquid nitrogen dewer</p>
</li>
<li>
<p>dry-ice bucket with samples</p>
</li>
<li>
<p>mortars & pestles</p>
</li>
<li>
<p>Mortexer</p>
</li>
</ul>
<p>Purification Station</p>
<ul>
<li>centrifuge</li>
<li>microcentrifuge tube racks</li>
</ul>
<h3 id="label-tubes">Label Tubes</h3>
<p>The samples originate from their 1.5mL cryo-vials, which are labelled with their cryo_id. Since it’s important to keep track of which samples were extracted using the same kit, the same reagents, the same day, etc. for batch effects, I use extraction IDs (extr1, extr2, extr3 , etc.) to label samples that were processed together.</p>
<p>Each sample will need the following 7 tubes labelled:</p>
<p><img src="%7B%7B%20site.url%20%7D%7D%7B%7B%20site.baseurl%20%7D%7D/assets/images/label-tube-diagram.jpeg" alt="label-7-tubes-diagram" /></p>
<p>You can also label an additional qubit tube if you are moving straight from extraction to quantification.</p>
<blockquote>
<p>Only thin-wall, clear 0.5 mL PCR tubes are appropriate for use in the Qubit® fluorometer. Acceptable tubes include <a href="https://www.thermofisher.com/order/catalog/product/Q32856?gclid=CjwKCAjwuqiiBhBtEiwATgvixAYKxw1FwdqIxIiPSxZ-ngW738s6bl94L2cdLCFjXfosHgMX1hyAShoCv9cQAvD_BwE&ef_id=CjwKCAjwuqiiBhBtEiwATgvixAYKxw1FwdqIxIiPSxZ-ngW738s6bl94L2cdLCFjXfosHgMX1hyAShoCv9cQAvD_BwE:G:s&s_kwcid=AL!3652!3!529745253294!p!!g!!qubit%20tubes!7851124045!80648205054&cid=bid_pca_aqb_r01_co_cp1359_pjt0000_bid00000_0se_gaw_bt_pur_con"><strong>Qubit® assay tubes</strong></a> <strong>(Invitrogen Cat.</strong> <strong>no.</strong> <strong>Q32856, 500 tubes) or <a href="https://us.vwr.com/store/product?keyword=10011-830">Axygen PCR-05-C tubes</a>(VWR, part number 10011-830)</strong>.</p>
</blockquote>
<p>The qubit tube should only be labelled on the top; the sides should be clear so that the qubit fluorescence can be read without impedance.</p>
<p>The intermediate tubes and qubit tubes should be labelled with the <code class="language-plaintext highlighter-rouge">cryo_id</code> & <code class="language-plaintext highlighter-rouge">extraction_id</code> , such as: <code class="language-plaintext highlighter-rouge">1Ea extr1</code>.</p>
<p>The <strong>FINAL</strong> tubes should be labelled with <code class="language-plaintext highlighter-rouge">cryo_id</code>, <code class="language-plaintext highlighter-rouge">DNA/RNA</code>, <code class="language-plaintext highlighter-rouge">extraction_id</code>, <code class="language-plaintext highlighter-rouge">date (ddMMMyy)</code>, & <code class="language-plaintext highlighter-rouge">initials</code>, such as:</p>
<div class="language-plaintext highlighter-rouge"><div class="highlight"><pre class="highlight"><code>1Ea
RNA
extr1
10APR23
SST
</code></pre></div></div>
<p>⚠️<strong>Important Notes! ⚠️</strong></p>
<ul>
<li><strong>Use <a href="https://www.amazon.com/dp/B09L3Q99WL/ref=sspa_dk_hqp_detail_aax_0?psc=1&sp_csd=d2lkZ2V0TmFtZT1zcF9ocXBfc2hhcmVk&spLa=ZW5jcnlwdGVkUXVhbGlmaWVyPUEyWUpaVTc1M0pRMVImZW5jcnlwdGVkSWQ9QTAxMTgzODBJOFI3QlZFQ0pKQUsmZW5jcnlwdGVkQWRJZD1BMDc2MTE0M1ExRUdBSkEwOFBBRiZ3aWRnZXROYW1lPXNwX2hxcF9zaGFyZWQmYWN0aW9uPWNsaWNrUmVkaXJlY3QmZG9Ob3RMb2dDbGljaz10cnVl">ethanol-proof lab markers</a> to label tubes</strong> <em>(ethanol is added to the green spin away collection tube)</em></li>
<li><strong>Label collection tubes, not filters!</strong></li>
<li><strong>Always wear lab gloves that have been sterilized before handling tubes!</strong></li>
<li><strong>Shake tubes out of their bags onto sterilized surface, don’t ‘reach in’ (this reduces potential contamination)</strong></li>
</ul>
<h3 id="prepare-buffers">Prepare Buffers</h3>
<ul class="task-list">
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />If using the 50-prep kit (D7003), add <code class="language-plaintext highlighter-rouge">96 ml 100% ethanol (104 ml 95% ethanol)</code> to the 24 ml DNA/RNA Wash Buffer concentrate.</p>
<p> <em>If using the 10-prep kit, DNA/RNA Wash Buffer (D7003T) is supplied ready-to-use and does not require the addition of ethanol.</em></p>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Reconstitute <code class="language-plaintext highlighter-rouge">lyophilized DNase I</code> with <code class="language-plaintext highlighter-rouge">DNase/RNase-Free Water</code> and mix by gentle inversion. Use immediately or store frozen aliquots.</p>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />50-prep, add 275 µl water</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />10-prep, add 55 µl water</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />Reconstitute <code class="language-plaintext highlighter-rouge">lyophilized Proteinase K</code> at 20 mg/ml with <code class="language-plaintext highlighter-rouge">Proteinase K Storage Buffer</code> and mix by vortexing. Use immediately or store frozen aliquots.</p>
<ul class="task-list">
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />50-prep, (60 mg), add 3.12 ml buffer</li>
<li class="task-list-item"><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />10-prep, (5 mg), add 0.26 ml (260 ul) buffer</li>
</ul>
</li>
<li class="task-list-item">
<p><input type="checkbox" class="task-list-item-checkbox" disabled="disabled" />To prepare a 1X solution of <code class="language-plaintext highlighter-rouge">DNA/RNA Shield™</code>, add an equal volume (5mL for the whole solution) of <code class="language-plaintext highlighter-rouge">nuclease-free water</code> (not provided) to the <code class="language-plaintext highlighter-rouge">DNA/RNA Shield™</code> (2X concentrate) (1:1) and mix with a quick pulse on the vortexer.</p>
</li>
</ul>
<h2 id="extraction-steps">Extraction Steps</h2>
<h3 id="grindhomogenize-mortar--pestle-samples">Grind/Homogenize: Mortar & Pestle Samples</h3>
<ol>
<li>
<p>Take <a href="#dry-ice">dry-ice</a> cooler to -80 freezer, pull out frozen samples and place them in the dry-ice cooler. Work quickly and carefully to sort through the vials and select the ones that you are working with. Nest the selected sample vials in the dry ice, and return the rest back to the -80 freezer. Bring working samples on dry-ice back to the lab bench.</p>
</li>
<li>
<p>Don cryo-gloves over nitrile gloves and ⚠️carefully⚠️ dispense a small amount (no more than 1L) of liquid nitrogen (LN2) into the transfer thermos. <strong>Everyone working with LN2 should have taken the Liquid Nitrogen Online Safety Course found <a href="https://www.ehs.washington.edu/training/liquid-nitrogen-safety">HERE</a></strong></p>
</li>
<li>
<p>Grind each sample with mortar & pestle on LN2 -</p>
<ol>
<li>⚠️carefully⚠️ pour a small amount of LN2 into a <a href="#sterilize">sterilized</a> mortar</li>
<li>Using <a href="#sterilize">sterilized</a> forceps, pluck out coral fragments from the cryo vial and place in the mortar until you have as much material as about the size of an M&M (roughly a 16mm diameter sphere). Material amount does not have to be precise.</li>
<li>Pestle the coral fragment until it is ground to a powder. Work quickly to ensure the sample remains frozen. Add more LN2 when it evaporates from the mortar. This step is challenging! LN2 evaporates very quickly and must be replenished multiple times.</li>
</ol>
</li>
</ol>
<p><img src="https://64.media.tumblr.com/tumblr_lzjqxk7t0K1qjhjdwo1_500.gifv" alt="pirates-jack-sparrow-in-davy-jones-locker-with-a-single-peanut" /></p>
<p>The coral is also very hard and prone to ‘squirting out’ from under the pestle, much like the gif of the peanut above!
{.notice: }</p>
<h3 id="lyse-bead-bash-samples">Lyse: Bead-Bash Samples</h3>
<ol>
<li>
<p>Use <a href="#sterilize">sterilized</a> scoopula to transfer the sample powder to its correspondingly labelled <a href="https://www.zymoresearch.com/collections/lysis-tubes/products/zr-bashingbead-lysis-tubes-0-1-0-5-mm">bead bashing tube</a></p>
</li>
<li>
<p>Aim to transfer 500uL of volume to each tube</p>
</li>
<li>
<p>Add 500uL of <code class="language-plaintext highlighter-rouge">DNA/RNA Shield</code> to each 2mL (0.1 - 0.5mm) bead-bashing tube with powdered sample and vortex to ensure powder is submersed in <code class="language-plaintext highlighter-rouge">DNA/RNA Shield</code></p>
</li>
<li>
<p>Set bead-bashing tubes in Mortexer and vortex at high speed for 40 minutes</p>
</li>
</ol>
<h3 id="proteinase-k-digestion">Proteinase-K Digestion</h3>
<ol>
<li>
<p>After bead bashing tubes are <a href="https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/Larvae-Ex-Protocol/#:~:text=Tubes%20after%20vortexing%20are%20intensely%20bubbly">‘intensely bubbly’</a></p>
</li>
<li>
<p>To tamp down bubbles, centrifuge in the mini-centrifuge for 1-2 minutes</p>
</li>
<li>
<p>Add Proteinase K & Buffer</p>
<ul>
<li>
<p>Add the appropriate volume of Pro K buffer and Proteinase K (Proteinase K is stored in the -20 after being reconstituted)</p>
</li>
<li>
<p>(10:1 ratio of sample:digestion buffer) & (2:1 ratio of digestion buffer:Proteinase K)</p>
</li>
<li>
<p>For tubes with 500ul add:</p>
<ul>
<li>50ul pro k buffer</li>
<li>25ul proteinase K</li>
</ul>
</li>
<li>
<p>If you have tubes with a larger volume you can scale up the volumes (ex for 700ul, you would use 70ul buffer and 35ul pro k)</p>
</li>
</ul>
</li>
<li>
<p>Let incubate at room temperature for 2 hours</p>
</li>
</ol>
<p>OR.. alternate protocol according to Putnam Lab Members <a href="https://kevinhwong1.github.io/KevinHWong_Notebook/20201027-DNA-RNA-Extractions-Porites-July-Bleaching-Experiment/">Kevin Wong Porites Extractions</a> and <a href="https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/porites-zymo-rna/">M.E. Schedl’s Porites Extractions</a>…</p>
<blockquote>
<ol>
<li>Add 500uL of RNA/DNA shield into each bead bashing tube</li>
<li>Using sterile forceps, add 0.25mm of coral fragment directly to bead bashing tube</li>
<li>Vortex at max speed for 2 minutes</li>
<li>Remove 400 μl of the supernatant and transfer to a new centrifuge tube</li>
<li>Centrifuge at 9000 rcf for 3 minutes</li>
<li>Transfer 300 μl supernatant to a new centrifuge tube and discard the old tube with pellet</li>
<li>Add 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and > 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample</li>
<li>Vortex to spin down</li>
<li>Add 345 μl of lysis buffer</li>
</ol>
</blockquote>
<p>[some <em>citrine</em> text]{style=”color:#E4D00A”} [some <em>pistachio</em> text]{style=”color:#93C572”}</p>
<p>[This text is highlighted in citrine.]{style=”background-color:#E4D00A”} [This text is highlighted in pistachio.]{style=”background-color:#93C572”}</p>
<h3 id="purify-dna--rna">Purify DNA & RNA</h3>
<h2 id="end-products">End Products</h2>
<p>The end products are two 1.5mL vials per sample that contain:</p>
<ol>
<li>100ul of DNA in nuclease-free water</li>
<li>100ul of RNA in nuclease-free water.</li>
</ol>
<p>Place these vials on ice and for RNA proceed with <a href="https://sarahtanja.github.io/lab-book/protocols/protocol-qbit/">Qubit RNA Broad Range Protocol</a> , for DNA proceed with Nanodrop DNA</p>
<p>OR, to continue lab-work later,</p>
<p>Place them in a wax freezer box, label the box, and freeze them in the -80C.</p>Sarah S.D. TanjaThe aim of this protocol is to take coral fragments that have been snap-frozen in liquid nitrogen and extract both DNA for downstream archaea & bacteria microbiome 16S Microbiome Sequencing and RNA for downstream coral-host tissue Tag-seq or RNA-seq analysis.