GSM6755918: Adult_Sperm_Bulk_WGBS; Trichosurus vulpecula; Bisulfite-Seq
Sperm were isolated from the epididymis of an adult possum by allowing sperm to diffuse out into warm PBS (37 °C) for 1 hour. Plasma was removed twice with PBS using centrifugation at 1,500 rpm for 2 min. The resulting pellet was resuspended in PBS and passed through a 20 µm cell strainer and stored in PBS at -80 °C. DNA was extracted fromsperm using a Bio-On-Magnetic-Beads (BOMB) protocol that utilises solid-phase reversible immobilization (SPRI) carboxyl-coated magnetic beads to isolate DNA (Oberacker et al., 2019; PMID: 30629605), with the addtion of 10% v/v ß-mercaptoethanol added to the cell lysate as a reducing agent (Fujikura et al 2017; PMID: 31453223) Bisulfite-converted genomic libraries from sperm DNA were prepared using a modified PBAT method outlined by Ortega-Recalde et al., 2021 (PMID:34009607)