Samples

4L14F, 5CL14F, 6L14HF = 456L14
4M14F, 5M14F, 6M14F = 456M14
7C4F, 8C4F, 9C4F = 789C4
7H9F, 8H9F, 9H9F = 789H9
8C14F, 9C14F = 89C14F¹
10M4F, 11M4F, 12M4F = 101112M4
10H4F, 11H4F, 12H4F = 101112H4
10M9F, 11M9F, 12M9F = 101112M9
13H14F, 14H14F, 15H14F = 131415H14
13L9F, 14L9F, 15L9F = 131415L9

¹ No 7C14F sample

Summary

Note

Overall success! Even in 89C14, which pooled 2 crosses instead of 3. Here I added extra DNA/RNA Shield to 89C14 to make the volume in the Zymo BashingBead for all samples uniform. AND despite forgetting to first run DNA/RNA Wash Buffer through the RNA filter prior to DNase treatment.

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
- 89C14 I ‘topped up’ with DNA/RNA Shield so that it had 1.5mL volume same as the other samples
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
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
Add 530uL of DNA/RNA Lysis Buffer to each tube ( a total of 1060uL volume in each 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). This will take 3 passes.
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 2.1mL + ethanol 2.1mL = 4.2mL
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

Warning

Forgot to do DNA/RNA Wash 400uL prior to DNase Treatment :(

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: 96.18

standard 2: 1912.96

RunID: 2024-08-24_xxxxxx²

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
456L14	5.20 ng/uL	5.22 ng/uL
456M14	5.00 ng/uL	5.00 ng/uL
789C4	7.52 ng/uL	7.60 ng/uL
789H9	7.70 ng/uL	7.76 ng/uL
89C14	5.06 ng/uL	5.02 ng/uL
101112M4	12.5 ng/uL	12.6 ng/uL
101112H4	8.00 ng/uL	8.10 ng/uL
101112M9	7.60 ng/uL	7.62 ng/uL
131415H14	6.12 ng/uL	6.22 ng/uL
131415L9	9.70 ng/uL	9.70 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.