Re-extracting low-yield sperm samples

Earlier I identified that some extractions did not yield enough DNA or RNA for libraries. Today, I extracted DNA and RNA from low pCO₂ samples 7 and 57, and high pCO₂ samples 48 and 64.

Methods: Sample Preparation

Step 1: Prepare for extractions.

• Label 2 sets of tubes RNase-free centrifuge tubes per sample: one for final RNA storage, and one for final DNA storage.
• Label spin columns
• Obtain samples from -80ºC freezer
• Placed the nuclease-free water on a heat block at 95ºC
• Obtained DNase I from the -20ºC freezer

Step 2: Obtain 50 µL of sperm sample in a seawater solution and pipet into a labelled centrifuge tube.

• Since the shipping tubes had 50 µL of left, I didn’t need to transfer it to a separate tube

Step 3: Add 4 volumes of DNA/RNA Lysis Buffer and mix

• I added 200 µL of Lysis Buffer to each sample for a total volume of 250 µL
• Vortexed for 10 pulses

Methods: Sample Purification

Step 4: Transfer the sample into a IC-MX spin column in a collection tube and centrifuge.

• These reagents are really soapy, so I learned that if I pipetted slowly I could avoid creating “soap” bubbles in the sample
• Kept the labelled spin columns for DNA extractions
• Saved the flow-through for RNA extractions

Step 5: For RNA only, add an equal volume of 95-100% ethanol to the flow-through and mix by pipetting. Transfer the flow-through into a new IC spin column in a clean collection tube. Centrifuge and discard the flow-through.

• Added 250 µL of EtOH and mixed

Step 6: For the RNA IC spin columns, perform a DNAse I treatment

• Added 400 µL of the DNA/RNA Wash Buffer to each column and centrifuged, then discarded the flow-through.
  • At this point, I continued with Step 7 for the DNA samples
• Made a DNase REaction Mix with 5 µL of DNase I and 35 µL DNA Digestion Buffer for each sample.
• Added the mix to the column matrix and incubated it at room temperature for 15 minutes before proceeding.
  • While the RNA samples were incubating, I finished Steps 8-10 with the DNA samples

Step 7: Add 400 µL DNA/RNA Prep Buffer to the column. Centrifuge and discard the flow-through.

Step 8: Add 700 µL DNA/RNA Wash Buffer to the column. Centrifuge and discard the flow-through.

Step 9: Add 400 µL DNA/RNA Wash Buffer to the column. Centrifuge at 16,000 rcf for 2 minutes. Transfer the column to a new microcentrifuge tube.

Step 10: Add 15 µL DNase/RNase-Free Water to the column. Incubate at room temperature for 5 minutes. Centrifuge to elute the DNA and RNA.

• Added 15 µL of 95ºC nuclease-free water to the column
• Samples were centrifuged for 1 minute

Methods: Sample Quantification

Step 11: Prepare the master solution using a 1:200 ratio of buffer to dye. For each sample and both standards, 200 µL of master solution is needed.

• RNA: I made 1600 µL of master solution using 8 µL of dye and 1592 µL of buffer. This would suffice for 8 samples (2 extra sample volumes)
• DNA: Pre-mixed master solution for the Qubit dsDNA HS kit (aka my favorite thing)

Step 12: For each standard’s designated Qubit assay tube, add 10 µL of the correct standard and 190 µL of the master solution.

Step 13: For each sample’s designated Qubit assay tube, add 1 µL of the sample and 199 µL of the master solution.

Step 14: Vortex all Qubit assay tube for 2-3 seconds at maximum speed. Incubate tubes at room temperature for two minutes.

Step 15: Use Qubit to quantify RNA and DNA yield in each sample tube.

• Updated yields can be found here
• I didn’t get any RNA yield from sample 57! For 57, my total DNA is ~100 ng, so that’s definitely not enough for a WGBS library. It’s likely that I won’t be able to sequence 57, which is unfortunate because it’s one of the few low pCO₂ samples from the experiment.
• I also didn’t get any RNA from 64, but this time I got ~200 ng RNA from 48. Depending on how much RNA is needed for RNA-Seq and small RNA sequencing, these samples may be viable. At least I have enough DNA from both samples for WGBS.
• Since I won’t be able to test ATAC-Seq on samples in seawater, I think I’m going to go ahead and extract the remaining samples while I wait for sequencing quotes from Zymo

Going forward

1. Talk to Zymo rep about input quantity needed for RNA-Seq
2. Re-extract male samples and check yields
3. Consolidate sample tubes and update concentrations
4. Send DNA and RNA for library preparation and sequencing