GSM8564759: atac_smarca5_ko2; Mus musculus; ATAC-seq
A polypropylene collection tube was coated with 3 ml Collection Buffer (20% FBS in 1X PBS) for 2 hours at RT with end-over-end mixing. Seminiferous tubules were mechanically isolated from the tunica and transferred to a 15ml conical tube containing 5ml Digestion Solution I [0.75 mg/ml collagenase type IV (Gibco, 17104-019) in Dulbecco's modified Eagle's media (DMEM, Gibco, 11965092) supplemented with 1:1000 with 1 mg/ml DNase I (StemCell Technologies, 07900)] warmed to 37 °C, followed by incubation at 37 °C for 10 mins with end-over-end rotation. 5ml of cold DMEM was added and tubules allowed to settle on ice. The supernatant was discarded and the tubules were washed again with 5ml cold DMEM to deplete Leydig cells. 5ml of DMEM was added followed by centrifugation at 500xg for 5 mins at 4°C. The supernatant was removed and 5ml of Digestion Solution II [Accutase (Gibco, A1110501) supplemented with 1 mg/ml DNase I at 1:1000 at room temperature)] was added to the tubules followed by incubation with end-over-end rotation for 10 mins at RT with gentle pipetting after 5 mins to disperse the tubules. 5ml of cold Complete Media (10% FBS in DMEM) was added and the sample was centrifuged at 500 xg for 5 mins at 4 °C. The cell pellet was then resuspended in 5 ml of cold Complete Medium, filtered through a 100 μm strainer, and the cell suspension was transferred to a fresh 15 ml conical tube and centrifuged at 500 xg for 5 mins at 4 °C.Cells were stained with Hoechst dye and isolated by flow cytometry based on 4C DNA content.  50,000 sorted pachytene or pachytene-like cells were processed for each ATAC-seq library using the Active Motif kit (53150) as per the manufacturer's protocol. 50,000 sorted cKIT+ cells were processed for each ATAC-seq library using the Active Motif kit (53150) as per the manufacturer's protocol.