GSM7509606: ChIP_HEK293T_Input_3; Homo sapiens; ChIP-Seq
HEK293T cells were washed twice with PBS and crosslinked with 1% formaldehyde (Pierce) for 10 min at room temperature. Crosslinking was quenched with 125 mM (final) glycine for 5 min and washed twice with PBS. Cells were lysed in 4 ml lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), 1x Protease inhibitor (Roche) for 10 min on ice. Lysed cells were centrifuged at 4,000 rpm for 5 min at 4°C and washed twice with 4 ml cold wash buffer (10 mM Tris-Cl pH 7.5, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0), 1x protease inhibitors. The pellet was resuspended in 1 ml shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris pH 7.5), 1x protease inhibitor and sheared on Covaris S220 (140 W, 5% duty factor, 200 bursts per cycle, 4°C) for 12 min (determined by time course optimization experiment). Extract was diluted with Triton X-100 (1% final) and NaCl (150 mM final) and cleared by centrifugation at 21,000g, 4°C for 10 min. For input, 10% of material was set aside. Cleared extract was supplemented with 2 μL of antibody and 75 nM competitor peptide and incubated overnight at 4°C. Immuno-precipitated chromatin was captured on 30 μl of Dynabeads protein G (Invitrogen) at 4°C for 1.5 hours. Beads were washed twice with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM HEPES–potassium hydroxide pH 7.5, 150 mM NaCl), twice with high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM HEPES–potassium hydroxide pH 7.5, 500 mM NaCl), once with LiCl Buffer (100 mM Tris-HCl pH 7.5, 0.5 M LiCl, 1% NP-40, 1% sodium deoxycholate) and once with TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). Enriched DNA was eluted in 100 μl of proteinase K buffer (20mM HEPES pH 7.5, 1 mM EDTA, 0.5% SDS) supplemented with 40 μg of proteinase K (Ambion) for 30 min at 50°C. Formaldehyde crosslinks were reversed by adding NaCl (150 mM final) and 0.25 μg DNase-free RNase (Roche), followed by incubation overnight at 65°C. DNA was isolated using QIAquick PCR purification kit (QIAGEN). ChIP-seq libraries were prepared by the Yale Center for Genome Analysis.