GSM7663843: HBL1_OCT2_proteintech_ChIP; Homo sapiens; ChIP-Seq
For each ChIP, 20 million cells were used. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature with agitation and quenched with 0.125 M glycine for 5 min at room temperature. After washing twice with PBS, cells were incubated in 1 ml cell lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40 with protease inhibitor) for 15 min on ice. Nuclei were isolated by centrifugation at 600×g for 30 s, resuspended in 1 ml nuclear lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS with protease inhibitor) and sonicated using a Bioruptor Pico (Diagenode) (30s on/off, 10 cycles). In all cases, the chromatin was centrifuged at maximum speed in a tabletop centrifuge for 15 min at 4 °C. The supernatant was mixed with 7 ml IP dilution buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 150  mM NaCl, 1% Triton X-100), and incubated with the 2.5 µg of the indicated antibody for 2 h. Protein A/G magnetic beads (25 µl, Dynabeads, Thermo Fisher Scientific) were washed twice with PBS and added to the antibody–chromatin mixture at 4 °C overnight. The beads were washed once with IP wash 1 buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash 2 buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate) and twice with TE (pH 8.0). Chromatin DNA was eluted, and cross-linking was reversed in 200 µl nuclear extraction buffer with 12 µl of 5 M NaCl and 1 µg /mL RNase A at 65 °C overnight. The beads were then discarded. The DNA-containing supernatant was treated with 4 µg /mL proteinase K at 56 °C for 20 min and purified using the QIAquick PCR purification kit (QIAGEN) in 60 μl water.