GSM6755889: 250822_PY02_Somatic; Trichosurus vulpecula; Bisulfite-Seq
Gonads were collected from male pouch young aged between 7 and 106 days postpartum (n=17). Germ cells were isolated from gonads using the isolation protocol outlined in Suzuki et al (2013; PMID: 23732002). The resulting pellet was resuspended in Zombie Near-Infra Red Viability Dye (BioLegend) and incubated at room temperature for 20 min. The lysate was washed in 5 % FBS (in PBS), then resuspended in SSEA1 monoclonal antibody (Invitrogen, MC-480) conjugated to DyLight 488 (1:300 in PBS/5% FBS; Invitrogen, MA1-022-D488) on ice for 30 min. The lysate was then washed, resuspended in sort buffer (5% FBS, 0.5 mM EDTA in PBS) and kept on ice prior to cell sorting. Germ cells were isolated from the cell lysate using a BD FACSAria Fusion which detects GFP using a 488 argon laser (BD Biosciences). A gonadal cell profile was produced using forward scatter (FSC) and side scatter (SSC) and was gated to exclude doublets and dead cells. GFP-negative cells (somatic cells) were sorted as negative controls into 0.2 % Triton-X and stored at -80 °C. Bisulfite conversion and library preparation from low cell input followed the single cell bisulfite sequencing protocol by Clark et al., 2017 (PMID: 28182018)