Using PAXgene tissue DNIA kit Processing 4 extra samples: 2 @ low pH, 2 @ ambient pH Following protocol. Note, I used the Tissue Tearer for homogenization.

Selected 4 samples from HL, 2 male + 2 female

• HL-6-18, Female, Cassette #12, tissue mass ~17.2ug
• HL-6-20, Male, Cassette #12, tissue mass ~11.2ug
• HL-6-17, Male, Cassette #12, tissue mass ~12.7ug
• HL-6-10, Female, Cassette #2, tissue mass ~10.5ug

Reviewed slides corresponding to these tissues under scope to identify location of gonad.

Used razor blad to cut gonad out of paraffin blocks. Identified area with gonad tissue using microscope slides, did my best to only cut out gonad tissue, but this is difficult b/c gonad is such a small area. I’m concerned that I’m unable to be confident in isolating only gonad tissue - it’s likely to be contaminated with gill, mantle, digestive gland, etc. I could pull DNA from the section on on the slide; I wouldn’t get much DNA but perhaps it would be enough and I could be confident in the tissute type, which I presume is very important in methylation analysis, as it likely differs by tissue.

Weighed collection tubes pre- and post- tissue to estimate the tissue mass I extracted from each block. (Goal is 10-20mg tissue, no more than 20mg.)

After removing ethanol supernatant (after xylene, pre TD1 buffer), incubated at room temp for ~10 mins, then 37C for ~15 minutes to evaporate ethanol fully.

Homogenized tissue in TD1 buffer with the Tissue Tearor for ~20 seconds at setting #15.

Opted to NOT add the RNA enzyme to digest RNA; therefore there should be RNA in my samples still.

Both 1-hr incubation periods extended to 1:15, at ~400 rpm (max RPM on Graham Young’s incubator/shaker). NOTE: Graham does have a thermomixer that achevies 1300rpm, but only 70C - I could use this for the 1st incubation, but need to determine whether there is a large enough tube holder - OR instead try using smaller tubes for the incubation steps (if I can).

HICCUP: Sometime during the 2nd incubation (at 80C) the shaker stopped shaking; the incubator likely busted a belt, as the motor sounds like it still works. So, the samples were incubated for 80C for 1:15, with unknown time at 400rpm (at most 45 minutes).

Did not have enought time to perform assay to quantify samples immediately after extration; eluted samples were stored in the refrigerator.