Subsampling remaining female tissue

Methods: Sample Preparation

Step 1: Prepare for extractions.

• Label 3 sets of tubes RNase-free centrifuge tubes per sample: one for frozen tissue, one for final RNA storage, and one for final DNA storage.
• Obtain samples from -80ºC freezer and place on wet ice
  • Since I only had four spatulas available to me, I decided to process samples in batches of 3-4. I grabbed one batch from the -80ºC at a time and covered the samples in wet ice to prevent samples from thawing too much.

Step 2: Cut and weigh no more than 10-15 mg of frozen tissue. Record weight of tissue used in extractions and place tissue in a new, labelled test tube.

• I tared the scale with a piece of weigh paper. I used a clean spatula with a sharp edge to cut the tissue in the tube. Once I got the weight I wanted, I transferred the tissue to the labelled centrifuge tube. The spatulas were washed in 200 mL of a 10% bleach solution, then rinsed with DI water after both samples were prepared.
• Some samples were very gamete rich (“milky”) so they were tricker to weigh. After getting the sample mass, I put the sample back in the tube the samples were mailed in so I could digest the milky gametes that I couldn’t put on the scale. I’ll need to transfer these samples to 1.5 mL centrifuge tubes after overnight incubation.

Table 1. Mass of samples used for DNA/RNA extractions. Samples that were too “milky” to transfer to 1.5 mL centrifuge tubes were kept in the mailing tubes after subsampling, meaning that I used all the sample in that tube. There were also samples that do not have any more remaining tissue after obtaining 10-15 mg of tissue.

Sample ID	Weight Sent (mg)	Subsample Mass (mg)	Notes
3	35	15	Milky
16	34	12.1
19	26	9.5	Milky
22	45	13.1
29	16	9.0	All sample used
35	45	12.7
39	35	10.1	Milky
41	18	11.5	All sample used
44	25	12.2
50	25	14.2	Milky
52	23	10.7
53	18	10.8	All sample used
76	22	~5-6	Very milky, couldn’t measure accurately
77	18	6.2	Milky

I put the subsampled and weighed tissues back in the -80ºC. Tomorrow, I’ll add DNA/RNA shield and digestion buffers to each sample and start an overnight incubation.

Going forward

1. Perform DNA and RNA extractions with subsampled tissues.
2. Check DNA and RNA yield and quality for female samples
3. Test extraction protocol for male samples
4. Send DNA and RNA for library preparation and sequencing