GSM6938448: DMRT1 Cut-And-Run d8 DAZL+ PGCLC replicate 2; Homo sapiens; OTHER
DZ+PGCLCs cells were bound to activated 10ul Concanavalin A -coated magnetic beads and incubated with DMRT1 antibody (Abcam, ab126741) or rabbit IgG(Cell Signaling, #2729) in Wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine and protease inhibitor) containing 0.1% Digitonin for 2hr at 4°C. After two washes, beads were incubated with Protein A/G-MNase fusion protein at 70 ng/mL in Digitonin-wash buffer for 1 hr at 4°C. After two washes, MNase activity was induced by addition of 3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.1% Digitonin and stopped by addition of 2X stop buffer (340 mM NaCl, 20 mM EDTA, 4mM EGTA, 0.1% Digitonin, RNAse A 100 ul/mL, Glycogen 50 ug/mL). Beads were incubated at 37°C for 30 min, supernatants were removed to a fresh tube and incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. DNA was extracted with phenol-chloroform extraction. Sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645S) for Illumina with KAPA HiFi™ Real-Time PCR Library Amplification Kit (Roche, KK2702)