Project Summary: 2022 RNA and DNA Extractions for Brown/Barott Pdam Project

This is the project summary for RNA and DNA extractions and RNA submission for the Pdam project in collaboration with the Barott Lab at UPenn (Dr. Kristen Brown and Katie Barott). There were 48 samples in total, and the metadata can be found here.

Outline of Content

1. Extractions
   • Protocol Link
   • 2022-10-27
   • 2022-11-02
   • 2022-11-08
   • 2022-11-11
   • 2022-11-18
2. Nanodrop
3. Plate Prep and Submission

Extractions

Extraction Protocol Link

2022-10-27: Extraction of 4 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Today I tested our protocol on these samples. I had to adjust the protocol slightly because they were preserved in RNAlater instead of DNA/RNA shield. I did the following:

  1. Thawed samples on ice.
  2. With sterilized forceps (10% bleach, DEPC water, 70% ethanol, air-dried), I removed one small (5mm x 5mm) fragment from the tube, in which most tubes had 5-6 small fragments of this size. I sterilized forceps between each sample.
  3. I briefly transferred this fragment to a clean 1.5 mL tube on ice to tap off any excess RNAlater and then immediately transferred the fragment to a 1.5 mL screw-cap tube with 800 uL DNA/RNA shield and 0.25 mL of 0.5mm glass beads.
  4. Bead beat for 1-2 minutes on the vortex at max speed. I started off with one minute for all samples but added a minute of bead-beating for samples if the liquid still looked very light-colored after one minute of homogenization.
  5. Briefly spin down and remove 400 uL of supernatant into a clean tube. Spin for 3 mins at 9,000 rcf.
  6. Put original samples and bead tubes back in -80 ºC freezer.
  7. Remove 300 uL of the supernatant into a new tube and continue on with the protocol below (from the Pro K step) as written.

• Other than the adjustments mentioned above, I followed protocol exactly.

• For my records: opened new kit today (Kit labelled 1/2 rec’d 10/4/22), used the columns, collection tubes, and DNA digestion buffer from this new kit.

• I bead-beat RS6D and RF25A for 2 mins instead of 1 min because they were still light-colored after one minute of bead-beating.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 183.61 (S1) & 22513.62 (S2)

RNA Standards: 416.40 (S1) & 9883.91 (S2)

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Next steps

Will use this protocol on the rest of the samples.

2022-11-02: Extraction of 12 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Continuting processing the Barott and Brown Pdam samples using the protocol detailed on 10/27/22.

• For my records: Took new Proteinase K, RNA/DNA Prep Buffer, and RNA/DNA Wash Buffer from the kit labelled 1/2 rec’d 10/4/22.

• See bead-beating notes below with Qubit values.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 104.76 (S1) & 23344.85 (S2)

RNA Standards: 417.37 (S1) & 9558.32 (S2)

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Note: Gel was only run for 40 minutes due to time constraints so is a little smushed-looking but all lanes look great!

2022-11-08: Extraction of 18 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Continuting processing the Barott and Brown Pdam samples using the protocol detailed on 10/27/22.

- Had much higher qubit values here because I started putting two of the tiny fragments into the bead beat tubes instead of one. Mostly this meant I only had to bead beat most samples for 1 minute instead of 2 to get a dark colored solution and resulted in great RNA! But I think the previous samples are great too, just slightly less in concentration.

• See bead-beating notes below with Qubit values.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 195.49 (S1) & 22843.75 (S2)

RNA Standards: 413.58 (S1) & 8989.92 (S2)

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

2022-11-11: Extraction of 14 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Continuting processing the Barott and Brown Pdam samples using the protocol detailed on 10/27/22.

- Had much higher qubit values here because I started putting two of the tiny fragments into the bead beat tubes instead of one. Mostly this meant I only had to bead beat most samples for 1 minute instead of 2 to get a dark colored solution and resulted in great RNA! But I think the previous samples are great too, just slightly less in concentration.

• See bead-beating notes below with Qubit values.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 191.69 (S1) & 22436.66 (S2)

RNA Standards: 422.67 (S1) & 9120.78 (S2)

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

And yes, we ran out of ladder. I think we have more but I could not find it.

Next steps

All done with these samples! Will check in with Hollie to see next steps for the extracted RNA and DNA. We could maybe redo some of the lower concentration extractions and add more tissue into the bead-beating steps for those.

I did re-QC the following five samples that had originally “nd” values for RNA quantity when measured using the RNA Broad Range Qubit kit. I thawed the RNA on ice and measured their RNA quantity using the Qubit RNA High Sensitivity kit, which uses different reagents and standards but the exact same protocol as the BR kit..

All of them look okay, but we could possibly redo the extractions with more tissue at the bead-beat step, especially for those with concentrations below 10.

2022-11-18: Re-Extraction of 12 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Continuting processing the Barott and Brown Pdam samples using the protocol detailed on 10/27/22.

• For these re-extractions, I had high-quality DNA and RNA for each sample but just not enough. I will proceed by adding 1-2 fragments depending on size to the original bead tube for each sample along with 400 uL of DNA/RNA shield that was previously removed. Then I will bead-beat as usual and continue as usual.

• See bead-beating notes below with Qubit values.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 199.11 (S1) & 22469.88 (S2)

RNA Standards: 421.54 (S1) & 9880.18 (S2)

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Dilutung new ladder: 4 uL water, 1 uL loading dye, 1 uL ladder

Next steps

All done with these samples!

QC of RNA for Brown/Barott Pdam Project, December 2, 2022

Nanodrop extracted RNA from the Barott and Brown Pdam extractions prior to sending the extracted RNA for sequencing. This is to test the purity of the extracted RNA. The blank used is the RNAse/DNAse free water included in the Zymo kit used for extracting the RNA.

Results

Protocol

The standard PPP Nanodrop protocol for RNA can be found here.

I used the nanodrop in the Lane lab with supervision/permission from Kristina Terpis, the Technician in the Putnam and Lane labs. The protocol is the same but it only can read one sample at a time and there is no printer, so I record the values by hand.

Prep and submit RNA for Brown/Barott Pdam Project, December 2, 2022

Dilute extracted RNA to 15 ug/uL in 30 uL and consolidate into one plate for RNAseq. This plate prep guide is based off of Dr. Kevin Wong’s protocol/example here.

Samples

Plate Map

Protocol

1) Remove samples from the -80 ºC freezer and place on a tube rack in an ice bucket to thaw.

2) Fill each well of the 96-well PCR plate with the corresponding diluent volume (I used the RNAse/DNAse free water included in the Zymo kit used for extracting the RNA).

3) Place plate onto a block frozen at -80 ºC.

4) Once samples are thawed, spun down, and pipette mixed the sample before adding the appropriate volume into the corresponding well.

5) Seal plate with aluminum foil tape and seal each well. Write on the label and the side of the plate with the following information:

- Date: 12/6/22
- Name: Kristen Brown
- Project: Heron Island Pdam
- Job Number: JA22512

6) Place remaining samples and plate back into the -80 ºC freezer.