1 Samples

1C14F, 2C14F, 3C14HF = 123C14
8M9F, 9M9F = 89M9¹
7H4F, 8H4F, 9H4F = 789H4
10L14F, 11L14F, 12L14F = 101112L14
10L4F, 11L4F, 12L4F = 101112L4
10L9F, 11L9F, 12L9F = 101112L9
13M4F, 14M4F, 15M4F = 131415M4
13H9F, 14H9F, 15H9F = 131415H9

¹ Sample 7M9F resulted in no surviving embryos and therefore we had no saved material for that vial

2 Summary

Note

Overall success! Decent Total RNA from all samples, even the one that was only pooled with 2 instead of 3 crosses. Sample 89M9 had a smaller starting volume in DNA/RNA Shield and therefore fewer passes through each of the DNA and RNA filters. Total process with 8 samples takes about ~5 hours of bench time.

3 Extraction Notes

This uses the Zymo Quick-DNA/RNA Miniprep Plus Kit and unless otherwise specified here, follows the M. Capitata Embryo DNA/RNA Extractions Protocol. All centrifugation ocurred at 14800rpm/16,162 xg.

Thaw samples to room temp
Transfer and pool samples to one Zymo BashingBead Tube
Homogenize for 10 minutes
- 10 min Zymo BashingBead Tube
  - The 4hpf samples are hardier and take a little longer to lyse than the other embryonic phases
Centrifuge for 1 min to get rid of bubbles
- Check for complete lysis of embryos
- Supernatent is clear!
Transfer 2 550uL aliquots of cleared supernatent to one new nuclease-free tube
- 89M9 had less volume available, transferred ~800uL total
Centrifuge for 1 min to pellet any beads
Split cleared supernatent into two aliquots of 530uL & transfer each aliquot to a new nuclease-free tube
- 89M9 had less volume, transferred ~700uL total
Add 530uL of DNA/RNA Lysis Buffer to each tube ( a total of 1060uL volume in each tube )
- 89M9 added 700uL of DNA/RNA Lysis Buffer, a total of 1400 in one tube
Vortex to mix for 5 seconds

—> Proceed to purification
Sequentially transfer each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s. SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into spare collection tube (15mL Falcon Tube)
Doubled volume of 100% (200 proof) ethanol to the RNA flow through in a falcon tube and vortexed to mix. End volume of flow through + ethanol = 4.2mL
- Except 89M9, end volume of flow through + ethanol = 2.8mL
Sequentially transfer RNA flow-through + ethanol in volumes =>700uL into the green Zymo Spin IIICG Column in a Collection Tube. This should take 6 ‘spins’
Saved 1st 2 spins (1400uL) for protein tube. Once collected place in -20 freezer.

Performed DNase I Treatment on green RNA spin column

DB = 75*10
print(DB)

[1] 750

Dnase = 5*10 
print(Dnase)

[1] 50

Note

    I didn't invert to mix... I always lose some volume 'beads' in the lid when I do this. Instead I flicked the tube to mix it and kept all the volume down in the conical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with!

400uL of Prep Buffer
700uL of DNA/RNA Wash Buffer
400uL of DNA/RNA Wash Buffer, 2min
Centrifuge 2min dry run
Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped 50uL of water directly over filter.

4 End Products

eluted DNA volume: 50uL in Zymo DNase-RNase Free Water

eluted RNA volume: 50uL in warmed (55C) Zymo DNase-RNase Free Water

Important

I upped the elution volume from 30 to 50 for RNA because I wanted to make sure the nuclease-free water volume was enough to saturate the whole filter and get the RNA from it… The minimum volume in the kit instructions is 50uL… And I do have an RNA Clean & Concentrator kit that allows me to reduce that if needed. As long as total RNA is at least 100ng, we’re good. That means that qubit value (ng/uL) times the elution volume.

5 Qubit QAQC

Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit.

Prepared working solution for 6 samples and two standards (plus some extra) in a 5mL tube:

buffer <- 199*15
print(buffer)

[1] 2985

reagent <- 1*15
print(reagent)

[1] 15

5.0.1 RNA

standard 1: 94.53

standard 2: 1590.87

RunID: 2024-08-22_062101²

² The Qubit date is ahead by 1 day (and I haven’t figured out how to set it straight yet)

sample_id	qubit_rna_1	qubit_rna_2
123C14	6.64 ng/uL	6.64 ng/uL
89M9	9.12 ng/uL	9.10 ng/uL
789H4	11.0 ng/uL	11.0 ng/uL
101112L14	7.22 ng/uL	7.08 ng/uL
101112L4	12.5 ng/uL	12.5 ng/uL
101112L9	11.4 ng/uL	11.4 ng/uL
131415M4	13.6 ng/uL	13.8 ng/uL
131415H9	10.6 ng/uL	10.5 ng/uL

6 Storage Location

RNA samples are stored in -80C ‘JPG Lab Thermo’ freezer on shelf 1 (top shelf) in coral-embryo-leachate RNA wax freezer boxes with green label tape.

DNA samples (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in coral-embryo-leachate DNA wax freezer box with yellow label tape.

Protein flow-through (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in coral-embryo-leachate PROTEIN wax freezer box with blue label tape.

--- title: "21-Aug-2024 Embryo RNA Extractions" author: "Sarah Tanja" date: 08/21/2024 date-format: long date-modified: today categories: [lab records] draft: false toc: true toc-exand: true toc-title: Contents <i class="bi bi-bookmark-heart"></i> toc-depth: 5 toc-location: left reference-location: margin citation-location: margin link-external-icon: true link-external-newwindow: true --- # Samples - 1C14F, 2C14F, 3C14HF = 123C14 - 8M9F, 9M9F = 89M9[^1] - 7H4F, 8H4F, 9H4F = 789H4 - 10L14F, 11L14F, 12L14F = 101112L14 - 10L4F, 11L4F, 12L4F = 101112L4 - 10L9F, 11L9F, 12L9F = 101112L9 - 13M4F, 14M4F, 15M4F = 131415M4 - 13H9F, 14H9F, 15H9F = 131415H9 [^1]: Sample 7M9F resulted in no surviving embryos and therefore we had no saved material for that vial # Summary ::: callout-note Overall success! Decent Total RNA from all samples, even the one that was only pooled with 2 instead of 3 crosses. Sample 89M9 had a smaller starting volume in DNA/RNA Shield and therefore fewer passes through each of the DNA and RNA filters. Total process with 8 samples takes about \~5 hours of bench time. ::: # Extraction Notes This uses the Zymo Quick-DNA/RNA Miniprep Plus Kit and unless otherwise specified here, follows the [M. Capitata Embryo DNA/RNA Extractions Protocol](https://sarahtanja.github.io/quarto-blog/posts/projects/coral-embryo-leachate/embryo-extractions.html). All centrifugation ocurred at 14800rpm/16,162 xg. - Thaw samples to room temp - Transfer and pool samples to one Zymo BashingBead Tube - Homogenize for 10 minutes - 10 min Zymo BashingBead Tube - The 4hpf samples are hardier and take a little longer to lyse than the other embryonic phases - Centrifuge for 1 min to get rid of bubbles - Check for complete lysis of embryos - Supernatent is clear! - Transfer 2 550uL aliquots of cleared supernatent to one new nuclease-free tube - 89M9 had less volume available, transferred \~800uL total - Centrifuge for 1 min to pellet any beads - Split cleared supernatent into two aliquots of 530uL & transfer each aliquot to a new nuclease-free tube - 89M9 had less volume, transferred \~700uL total - Add 530uL of DNA/RNA Lysis Buffer to each tube ( a total of 1060uL volume in each tube ) - 89M9 added 700uL of DNA/RNA Lysis Buffer, a total of 1400 in one tube - Vortex to mix for 5 seconds —\> Proceed to purification - Sequentially transfer each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s. **SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into spare collection tube (15mL Falcon Tube)** - Doubled volume of 100% (200 proof) ethanol to the RNA flow through in a falcon tube and vortexed to mix. End volume of flow through + ethanol = 4.2mL - Except 89M9, end volume of flow through + ethanol = 2.8mL - Sequentially transfer RNA flow-through + ethanol in volumes =\>700uL into the green Zymo Spin IIICG Column in a Collection Tube. This should take 6 ‘spins’ - Saved 1st 2 spins (1400uL) for protein tube. Once collected place in -20 freezer. - Performed DNase I Treatment on green RNA spin column ```{r} DB = 75*10 print(DB) Dnase = 5*10 print(Dnase) ``` ::: callout-note ``` I didn't invert to mix... I always lose some volume 'beads' in the lid when I do this. Instead I flicked the tube to mix it and kept all the volume down in the conical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with! ``` ::: - 400uL of Prep Buffer - 700uL of DNA/RNA Wash Buffer - 400uL of DNA/RNA Wash Buffer, 2min - Centrifuge 2min dry run - Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped 50uL of water directly over filter. # End Products eluted DNA volume: 50uL in `Zymo DNase-RNase Free Water` eluted RNA volume: 50uL in warmed (55C) `Zymo DNase-RNase Free Water` ::: callout-important I upped the elution volume from 30 to 50 for RNA because I wanted to make sure the nuclease-free water volume was enough to saturate the whole filter and get the RNA from it... The minimum volume in the kit instructions is 50uL... And I do have an RNA Clean & Concentrator kit that allows me to reduce that if needed. As long as total RNA is at least 100ng, we're good. That means that qubit value (ng/uL) times the elution volume. ::: # Qubit QAQC Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit. Prepared working solution for 6 samples and two standards (plus some extra) in a 5mL tube: ```{r} buffer <- 199*15 print(buffer) reagent <- 1*15 print(reagent) ``` ### RNA standard 1: 94.53 standard 2: 1590.87 RunID: 2024-08-22_062101[^2] [^2]: The Qubit date is ahead by 1 day (and I haven't figured out how to set it straight yet) +---------------+-------------+-------------+ | sample_id | qubit_rna_1 | qubit_rna_2 | +===============+=============+=============+ | - 123C14 | 6.64 ng/uL | 6.64 ng/uL | +---------------+-------------+-------------+ | - 89M9 | 9.12 ng/uL | 9.10 ng/uL | +---------------+-------------+-------------+ | - 789H4 | 11.0 ng/uL | 11.0 ng/uL | +---------------+-------------+-------------+ | - 101112L14 | 7.22 ng/uL | 7.08 ng/uL | +---------------+-------------+-------------+ | - 101112L4 | 12.5 ng/uL | 12.5 ng/uL | +---------------+-------------+-------------+ | - 101112L9 | 11.4 ng/uL | 11.4 ng/uL | +---------------+-------------+-------------+ | - 131415M4 | 13.6 ng/uL | 13.8 ng/uL | +---------------+-------------+-------------+ | - 131415H9 | 10.6 ng/uL | 10.5 ng/uL | +---------------+-------------+-------------+ # Storage Location RNA samples are stored in -80C 'JPG Lab Thermo' freezer on shelf 1 (top shelf) in `coral-embryo-leachate RNA` wax freezer boxes with green label tape. DNA samples (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in `coral-embryo-leachate DNA` wax freezer box with yellow label tape. Protein flow-through (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in `coral-embryo-leachate PROTEIN` wax freezer box with blue label tape.