GSM7439453
FACS purified, spermatogonia were attached to Poly-D-Lysine (R&D Systems, Cat# 3439-200-01) coated 24-well plates by 1 minute centrifugation at 80 × g (instead of binding to Concanavalin A beads). For H2AK119Ub1 Cut&Run, >100,000 c-Kit(+) spermatogonia were used per each replicate experiment. For CBX2 and RING1B Cut&Run, 20,000 THY1(+)&ITGa6(+) or c-Kit(+) spermatogonia were used for each replicate experiment. After the attachment, cells were fixed with 1% formaldehyde in PBS for 15 minutes. After two PBS washes and one PBST (0.05% Triton X-100 (v/v)) wash, cells were incubated with primary antibodies in PBST BSA (0.05% Triton X-100, 3% BSA) overnight at 4 ºC. Next day, cells were washed twice with PBST and finally washed with PBST EDTA (2 mM). Cells were then incubated with 500 ng/mL pA-MNase (protein A-MNase) in PBST BSA EDTA for more than 1 hour at 4 ºC. After pA-MNase binding, cells were washed with PBST EDTA twice, and the plate was placed on wet ice. Cells were then incubated in pre-chilled 200 µL PBST with 2 mM CaCl2 for 30 minutes. After MNase cutting, 200 µL stop solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/µL RNase A) was added, and the plate was placed in a 37 ºC oven for 15 minutes. Released cut fragments were de-crosslinked in 0.1% SDS and 125 µg/mL Proteinase K (Sigma-Aldrich, Cat# 03115828001) in a 65 ºC oven for >12 hours. DNA was extracted using QIAquick PCR Purification Kit (Qiagen, Cat# 28104). Illumina sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina kit (New England Biolabs, Cat# E7645) based on manufacturer's instructions. Adapters used in 0.6 µM working concentration. Final PCR cycles were between 9 (H2AK119Ub1, started from ~100,000 cells) to 11 (CBX2 and RING1B, started from ~20,000 cells). Number of PCR cycles were determined to be in the exponential phase by monitoring the reaction with SYBR green dye in a real time PCR machine.