WT_CTCF_Rep2
1% formaldehyde in PBS was used to crosslink the cells for 10 min, followed by quenched with 125mM glycine on ice. Cells were collected and flash frozen in liquid nitrogen, then stored at -80C for use. Frozen crosslinked cells were thawed on ice and then resuspended in lysis buffer I (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors). After rotated for 10 min at 4C, the cells were collected, and resuspended in lysis buffer II (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors). After rotated for 10 min at 4C, the cells were collected, and resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, and 1% Triton X-100, protease inhibitors) for sonication. Sonicated lysates were cleared once by centrifugation at 16,000 g for 10 min at 4C. Input material was reserved. The remainder was incubated with magnetic beads bound with antibody to enrich for DNA fragments overnight at 4C. Beads were washed with wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1mM EDTA pH 8.0, 0.7% Na-Deoxycholate, 1% NP-40) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl) in order. Beads were removed by incubation at 65C for 30 min in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). Cross-links were reversed overnight at 65C. To purify eluted DNA, 200 mL TE was added and then RNA was degraded by incubation in 8ul 10 mg/mL RNase A at 37C for 2h. Protein was degraded by addition of 4ul 20 mg/mL proteinase K and incubation at 55C for 2h. Phenol: chloroform: isoamyl alcohol extraction was performed followed by an ethanol precipitation. The DNA was then resuspended in 50ml TE and used for qPCR. For ChIP-qPCR experiments, qPCR was performed using SYBR qPCR Master mix (Enzyme, #Q711-00).