1 Samples

8C9F, 9C9F = 89C9¹
7H14F, 8H14F, 9H14F = 789H14
10M14F, 11M14F, 12M14F = 101112M14
13H4F, 14H4F, 15H4F = 131416H4
13L14F, 14L14F, 15L14F = 131415L14
4C4F, 5C4F, 6C4F = 456C4
1M14F, 3M14F = 13M14
4H14F, 5H14F = 45H14
7M4F, 8M4F, 9M4F = 789M4

¹ 7C9F used in Aug 12th test extraction

2 Summary

Note

Sample 789H14 extraction was ‘too low’ even for the High Sensitivity RNA Qubit Assay. I am considering shifting from Standard RNA-seq to ultra-low input RNA-seq… as the RNA quantity is ‘enough’ given the sample starting material, but consistently low.

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
- 13M14 had very small amount of starting material
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
```
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: 92.47

standard 2: 1889.51

RunID: 2024-08-27_054059²

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
89C9	9.04 ng/uL	9.06 ng/uL
789H14	too low	too low
101112M14	14.0 ng/uL	13.7 ng/uL
131415H4	17.4 ng/uL	17.3 ng/uL
131415L14	10.7 ng/uL	10.7 ng/uL
456C4	11.1 ng/uL	11.2 ng/uL
13M14	6.38 ng/uL	6.40 ng/uL
45H14	8.14 ng/uL	8.10 ng/uL
789M4	10.0 ng/uL	9.98 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: "26-Aug-2024 Embryo RNA Extractions" author: "Sarah Tanja" date: 08/26/2024 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 - 8C9F, 9C9F = 89C9[^1] - 7H14F, 8H14F, 9H14F = 789H14 - 10M14F, 11M14F, 12M14F = 101112M14 - 13H4F, 14H4F, 15H4F = 131416H4 - 13L14F, 14L14F, 15L14F = 131415L14 - 4C4F, 5C4F, 6C4F = 456C4 - 1M14F, 3M14F = 13M14 - 4H14F, 5H14F = 45H14 - 7M4F, 8M4F, 9M4F = 789M4 [^1]: 7C9F used in Aug 12th test extraction # Summary ::: callout-note Sample 789H14 extraction was 'too low' even for the High Sensitivity RNA Qubit Assay. I am considering shifting from Standard RNA-seq to ultra-low input RNA-seq... as the RNA quantity is 'enough' given the sample starting material, but consistently low. ::: # 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 - 13M14 had very small amount of starting material - 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 ```{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: 92.47 standard 2: 1889.51 RunID: 2024-08-27_054059[^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 | +===============+=============+=============+ | - 89C9 | 9.04 ng/uL | 9.06 ng/uL | +---------------+-------------+-------------+ | - 789H14 | too low | too low | +---------------+-------------+-------------+ | - 101112M14 | 14.0 ng/uL | 13.7 ng/uL | +---------------+-------------+-------------+ | - 131415H4 | 17.4 ng/uL | 17.3 ng/uL | +---------------+-------------+-------------+ | - 131415L14 | 10.7 ng/uL | 10.7 ng/uL | +---------------+-------------+-------------+ | - 456C4 | 11.1 ng/uL | 11.2 ng/uL | +---------------+-------------+-------------+ | - 13M14 | 6.38 ng/uL | 6.40 ng/uL | +---------------+-------------+-------------+ | - 45H14 | 8.14 ng/uL | 8.10 ng/uL | +---------------+-------------+-------------+ | - 789M4 | 10.0 ng/uL | 9.98 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.