RNA and DNA Extractions for ENCORE Project, October 24, 2022

Protocol Link

Extraction of 3 samples from ENCORE Project. These samples were collected in Bermuda in August 2022 for the ENCORE TPC project. There are 160 samples total from the following 4 species: Diploria labyrinthiformis (DLAB), Montastraea cavernosa (MCAV), Madracis decactis (MDEC), and Porites astreoides (PAST)

Samples

Today I am trying to homogenize the sample by bead-beating using Kevin Wong’s protocol here:

Sample Preparation and Digestion

1. Add 0.25 mL of glass beads (0.5mm) and add 500 μl of RNA/DNA shield into each empty centrifuge tube.
2. Take samples out from -80 °C on dry ice.
3. Using sterile clippers, add 0.25mm of tissue into the centrifuge tube with beads and RNA/DNA shield.
4. Vortex at max speed for 2 minutes.
5. Remove 400 μl of the supernatant and transfer to a new centrifuge tube.
6. Centrifuge at 9000 rcf for 3 minutes.
7. Transfer 300 μl supernatant to a new centrifuge tube and discard the pellet.
8. Add 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.
9. Vortex and spin down.
10. Add 345 μl of lysis buffer.
11. Continue with DNA and RNA extraction protocol

Notes

• Followed protocol exactly.

• I followed Kevin’s bead-beating protocol as written at the end of my last notebook entry. I added 0.25 mL of glass 0.5mm beads to a clean screw-top 1.5 mL tube and added one of the fragments (to the tube with none of the sample DNA/RNA shield and trying to minimize mucus carry-over) with 500 of clean DNA/RNA shield. Then, I bead-beat using a vortexer on high speed for 3 minutes. Then, I removed 400 uL of the supernatant into a clean 1.5 mL tube and spun down at 9,000 rcf for 3 minutes. Then, I removed 300 uL of the supernatant into a clean tube and proceeded with the ProK digestion as written in the protocol.

Qubit Results

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

DNA Standards: 189.35 (S1) & 23403.63 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Past-A5	Porites astreoides	22	5.06	4.88	4.97
Past-E2	Porites astreoides	40	10.5	10.2	10.35
Past-F6	Porites astreoides	26	5.88	5.74	5.81

RNA Standards: 419.78 (S1) & 9776.84 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Past-A5	Porites astreoides	22	nd	nd	0
Past-E2	Porites astreoides	40	nd	nd	0
Past-F6	Porites astreoides	26	nd	nd	0

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

Next steps

Yay. Still not working!

Some posibilities that are totally different approaches instead of tiny tweaks

• bead-beat in lysis buffer instead of DNA/RNA shield
  • Basing this off of the Zymo Quick RNA protocol (as opposed to the Quick DNA/RNA protocol) which suggests homogenizing tissue in Lysis buffer. I will take fragments out of the DNA/RNA shield, submerge in lysis buffer + beads, and then bead beat? Then will take 400 uL of the supernatant and skip the Pro K digestion.
• Alternatively, I could also increase the amount of lysis buffer. Instead of 1:1, I could do add 500 ul to the 345 uL of sample + proK mixture.
• According to Kevin, 55ºC heating is not recommended by Zymo anymore, so I will no longer be trying to optimize that step. Kevin recommends optimizing the centrifugation after ProK digestion to remove symbiont cells.