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

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.

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.

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.

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

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

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.