Final sperm sample extractions

Since saving cells in seawater can lead to lysis, there’s a small chance that these sperm cells would be viable for ATAC-Seq. Instead of saving the tissue samples, I decided to do one last round of DNA and RNA extraction! My DNA yields are pretty good and another round of DNA extractions wouldn’t hurt, so I was mainly focused on getting more RNA. Compared to the female samples, the sperm samples have lower RNA yields. This makes sense since eggs are filled with maternal RNA (and I had a lot more starting sample).

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.

• Grace came into lab to help out, so she did Steps 9-10 with the RNA samples!

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
• After we quantified everything, we consolidated all sample tubes and reorganized the box in the -80ºC so there was only one DNA tube and one RNA for each sample. The final yields are in this spreadsheet
  • While consolidated, I discarded tubes that had no RNA yield: 57-1, 57-2, and 64-2.I have a sinking feeling I discarded 48-1 (a tube that had RNA in it) instead of 57-1…I’ll check the box in the -80ºC and re-quantify yield tomorrow if needed.

Nothing to do now but wait for Zymo to get back to me and send the samples out!

Going forward

1. Send DNA and RNA for library preparation and sequencing