GSM5939935: Replicate 2 hypoxia input; 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). 5% of each lysate was used for input and the rest was used for IP. Elution buffer was added to input samples to match the volume of IP samples. DNA samples were treated by 0.1 mg/ml Proteinase K (Fisher Scientific, 25-530-049) for 4 hrs at 65°C in buffer containing 0.003% SDS, 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0). 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.