Chromosome axis protein SYCP2 recruits HORMAD2 to enable meiotic synapsis quality control in mice
Chromosome axis protein SYCP2 recruits HORMAD2 to enable meiotic synapsis quality control in mice
Testes were detunicated and seminiferous tubules were dissociated in 1 mL HBSS containing Collagenase A (25 mg/mL), Dispase II (25 mg/mL) and DNase I (2.5 mg/mL) for 30 min at 37 °C with intermittent mixing. Digestion was quenched with 4 mL DMEM + 10% FCS, cells were pelleted (400 rcf, 5 min, RT), washed in DPBS-S (PBS + 10% knockout serum), and filtered through a 40 µm mesh. For dead-cell removal, the DPBS-S wash and filtration step was repeated, followed by differential-speed centrifugation (400/200/100 rcf) to enrich for viable cells. Cells were incubated with anti-ACRV1-PE (30 min, RT), washed, incubated with anti-PE magnetic microbeads (30 min, RT), and subjected to negative magnetic selection on Miltenyi MS columns to collect the viable, unlabeled flow-through. After a final wash and pellet, cells were resuspended in PBS + 0.04% BSA and labelled with Cell Multiplexing Oligos (CMOs) according to the manufacturer's instructions. Dead cell removal was done on two WTs Nucleic acid extraction was performed as part of the library construction.