RNA Re-extractions for Time Series Project, September 9, 2025

Continuation of Natalie’s amazing work extracting these samples, latest post here

Protocol Link

Re-extraction of 2 Montipora capitata samples from the Time Series experiment done in June and July of 2025. These samples had less than 500 ng of total RNA and will be re-extracted with modified protocol to increase yield for sequencing.

I made several adjustments to the protocol to try to maximize yield and minimize mucus carryover.

Samples

The sample IDs are MON: RO_H1 & R24_H3

Notes

• For sample prep bead beat was used in the original tube and vortexed for an additional 1 minute.
• Took 2X volume out of the DNA/RNA shield tube to double biomass input into extraction (especially because the shield in these tubes has been refilled, so much of the tissue was already lysed into DNA/RNA shield that had been removed in previous extractions)
• 600 uL of new DNA/RNA shield was added to sample tubes after the 600 uL was taken out for extraction for storage.
• Adjusted protocol for 2X volume accordingly: added 60 uL ProK digestion buffer and 30 uL ProK
• Performed a heated proteinase K digestion after adding the above and inverting 3X.
  • Digested at 55 ºC on the thermomixer, at 300 rpm, for 10 minutes
• Then spun down to pellet debris for 10 minutes at 9,000 rcf, at 4 ºC
• Then moved supernatant to new tube and added equal volume DNA/RNA lysis buffer. This had to be run through the DNA column in two separate additions of 700 uL
• Flow through from each spin was kept and moved into a new 1.5 mL tube
• To each of those flow-through tubes, 700 uL ethanol was added (worked one tube at a time) and mixed thoroughly (pipette up and down 10 times)
• Then 700 uL of this mixture was added to the RNA column and spun
• Then repeated with the remaining 700 uL in that tube
• Then did the process again for the second 1.5 mL tube of flow through
• Rest of extraction performed as written (except changes below)
• Eluted RNA to 80 uL of RNAse/DNAse free water warmed to 37 ºC (2 additions of 40 uL)
  • After the second elution, the entire eluate was reapplied to the column to get out any last RNA
  • DNA was not extracted
• 2 uL were used for Qubit and 8 uL were used for gel

Qubit Results

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

HS RNA Standards: 44.68 (S1) and 939.13 (S2)

colony_id	Species	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
0 H1	Montipora capitata	3.19	3.16	3.18
24 H3	Montipora capitata	9.19	9.24	9.22

This 24 H3 extraction was used for sequencing.

• RNA samples in -80 freezer

DNA and RNA Quality Check gel

Did not run gel.