GSM7792023
Cells were cross-linked with 2 mM disuccinimidyl glutarate in PBS/1 mM MgCl2 for 40 min at room temperature followed by 1% formaldehyde in PBS for 15 min at room temperature. The reaction was stopped by 125 mM glycine. The cells were washed with PBS and stored at –80°C prior to use. The cells were lysed in Lysis buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroyl sarcosine) containing proteinase inhibitor cocktail (Roche). Sonication was conducted with the Handy sonicator UR-21P (Tomy) to generate DNA fragments of approximately 150-450 bp. The sonicated lysates from approximately 2 x 106 cells were diluted in ChIP dilution buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 1% IGEPAL-CA-630, 10% glycerol) containing proteinase inhibitor cocktail and incubated overnight at 4°C with 50 µl of protein G magnetic beads (Invitrogen) that were preincubated with ~2 µg of mouse Zscan4 antibodies. The precipitants were washed once with high salt wash buffer (20 mM Tris-HCl, pH8.0, 400 mM NaCl, 2 mM EDTA, 0.1% SDS, 0.2% Triton-X), three times with LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP40, 1% Na-deoxycholate) and once with 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 10 mM NaCl. Bound chromatin was eluted in elution buffer (90 mM NaHCO3, 1% SDS), followed by RNase treatment at 37°C for 30 min and crosslink reversal with decross-linking mixture (2 M NaCl, 0.1 M EDTA, 0.4 M Tris-HCl, pH 6.8) containing proteinase K at 65°C for 3 h. DNA was purified by a phenol-chloroform-isoamylalcohol extraction and ethanol precipitation. ChIP DNA libraries were prepared with the NEBNext ChIP-Seq Library Prep Kit for Illumina (NEB)