GSM5799585: SPG-WT1 (S9.6 CUT&Tag); Mus musculus; OTHER
After removal of the tunica albuginea, the testes were incubated in 5mL of DPBS containing 120 U/mL of collagenase type I at 32 °C with gentle agitation for 8~10 min until seminiferous tubules were dispersed, The dispersed seminiferous tubules were further digested with 5 ml 0.25% trypsin, plus 0.1 ml 5mg/ml DNase I at 32°C for 8 min. The digestion was terminated by adding 0.5 mL FBS to inactivate trypsin. The cell suspension was filtered through a 70 μm cell strainer and then centrifuged at 500 g for 5 min at 4 °C. The cells' pellet was resuspended at a concentration of 106 cells/ml in DMEM with Hoechst 33342 (5 μg/106 cells) and 5 μl DNase I, followed by cell suspension was rotated for 30min at 32 °C with gentle rotation. Centrifuged at 500 g for 5 min at 4 °C to remove supernatant. Immediately before sorting, the cells were stained with propidium iodide (1 μg/106 cells) at 25 °C and filtered with a 40 μm cell strainer. 5 × 10^4 spermatogonia were sorted and washed once in 0.5 ml wash buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, and 1× protease inhibitors], and were incubated with 10 μl pre-washed concanavalin A–coated magnetic beads in a 1.5 ml microtube at 25 °C for 10 min. Cells were resuspended in 80 μl ice-cold antibody incubation buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitors, 0.025% digitonin, 0.01% NP-40, and 2 mM EDTA] with S9.6 antibody (MABE1095, Millipore) at 1:100 dilution, and then rotated end-over-end at 4 °C overnight. Subsequently, cells were resuspended in 100 μl dig-wash buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitors, and 0.025% digitonin] with rabbit anti-mouse IgG (Ab6709, Thermo Fisher) at 1:100 dilution and rotated end-over-end at 25 °C for 1 h. After washing, home-made pA-Tn5 adapter complexes in dig-300 buffer [20 mM Hepes (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitors, and 0.01% digitonin] were added to samples at a final concentration of 1:100 and then rotated end-over-end at 25 °C for 1 h. After washing, cells were resuspended in 300 μl tagmentation buffer [dig-300 buffer with 10 mM MgCl2] and incubates at 37 °C for 1 h. To stop tagmentation and solubilize DNA fragments, 10 μl 0.5M EDTA, 3 μl 10% SDS and 2.5 μl 20 mg/mL Proteinase K were added to each sample and incubated at 50 °C for 1 h. After extraction with phenol-chloroform and ethanol precipitation, libraries were amplified by PCR. The barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform to generate 150 bp paired-end reads.