GSM5939931: Replicate 1 normoxia IP; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 10 min at 37 ℃ and then quenched with 125 mM glycine for 1 min. Cells were sheared in buffer containing: 0.1% SDS, 50 mM Tris-HCl (pH 7.6), 1 mM EDTA (pH8.0), 0.002% Triton X-100, supplemented with PMSF (Roche, 10837091001) and protease inhibitor (Roche, 11836170001). Lysates were sonicated with a Bioruptor for a total of 40 cycles (30 sec on/ 30 sec off each cycle). Sonicated lysate was incubated with HIF1ɑ antibody (Novus Biologicals, NB100-479) conjugated with Dynabeads Protein A (Life Technologies, 10001D) + G (Invitrogen, 10003D) overnight at 4 ℃. Beads were washed twice for 5 mins each, first with RIPA buffer (10mM Tris-HCl pH7.6, 1 mM EDTA, 0.1% SDS, 0.1% NaDOC, 1% Triton X-100), then RIPA buffer supplemented with 0.3M NaCl, LiCl buffer (0.21 M LiCl, 0.5% NP-40, 0.5% NaDOC) and finally TE buffer plus 0.2% Triton X-100. Next, beads were washed once with TE buffer for 5 mins. DNA samples were eluted from beads in buffer containing 0.003% SDS, 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0), 0.1 mg/ml Proteinase K (Fisher Scientific, 25-530-049) for 4 hrs at 65°C. ChIP-DNA was purified by AMPure XP (Beckman Coulter, A63881). RNA-seq and ChIP-seq libraries were prepared for sequencing by the University of Chicago Genomics Facility following standard protocols.