Whole genome bisulfite sequencing from four stages of zebrafish sperm development
Whole genome bisulfite sequencing from four stages of zebrafish sperm development
A pool of fresh testicular samples was dissected and disaggregated by a combination of mechanical and enzymatic methods. The resulting individualized cells were fixed in formaldehyde and then, sorted using a BD FACSDiscover S8 Cell Sorter. Cells were sorted according to their morphology, nucleus complexity and DNA content. Four different populations were sorted: spermatogonia, primary spermatocytes, round spermatids and sperm. Samples were dissolved in homogenization buffer (20 mM Tris pH 8.0, 100 mM NaCl, 15 mM EDTA, 1% SDS, 0.5 mg/ml Proteinase K) at 55⁰C. Two Phenol/Chlorophorm/Isoamylalcohol (25:24:1, PCI) extractions were performed. DNA was precipitated using 1/5 volume of 4 M NH4Ac and 2.5 volumes of ice-cold absolute ethanol, and 1uL of linear acrylamide. Samples were incubated overnight at -20⁰C. The DNA was pelleted and resuspended in nuclease free water. Bisulfite converted libraries were generated using the Pico Methyl-Seq Library Prep Kit (Zymo Research, Cat. D5456) following manufacturer's instructions.