GSM8528486: NIPBL-A2-dTAG-4hr_RAD21_input; Homo sapiens; ChIP-Seq
For HA ChIP-seq, cells were fixed with 2 mM DSG for 45 minutes, washed with DPBS, then fixed further 1% formaldehyde for 10 minutes. Formaldehyde was quenched with 125mM glycine for 10 minutes. For other ChIP-seq experiments, cells were fixed with 1% formaldehyde for 10 minutes, then quenched with 125mM glycine for 10 minutes. All cells were lysed using formaldehyde lysis buffer (FALB; 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with 1% SDS and 1X PIC, and sonicated for 9 minutes (10 seconds on/10 seconds off) at 25% power using an Active Motif Epishear Probe Sonicator. Antibodies were pre-bound to blocked Protein A Dynabeads for 6-8 hours at 4°C before being added to chromatin that had been diluted out 1:10 with FALB (no SDS), incuating overnight at 4°C. The following day, beads were washed as described above, then eluted for 1 hour at 65°C in 50 mM Tris pH 8, 1 mM EDTA, 1% SDS. Supernatant was transferred to a new tube, supplemented with 200 mM NaCl and proteinase K, then incubated for 30 minutes at 55°C followed by overnight at 65°C. The following day, samples were treated with RNAse for 2 hours at 37°C, before DNA was purified using Zymo ChIP DNA Clean & Concentrator kit (#D5205). For CTCF input and ChIP, samples were size-selected using AMPure beads and libraries prepared using 2S Plus DNA Library Kit (IDT #10009878). Otherwise, libraries were prepared using Diagenode Microplex v3 (#C05010001) and UDIs (Set I: # C05010008; Set 2: #C05010009).