ChIP protocol

ChIP Protocol

First, 5×107 (p300/RNAP2/MED1/PcG ChIPs) or 1×107 (histone ChIPs) cells were crosslinked with 1% formaldehyde for 10min at room temperature and quenched with 0.125M glycine for 10 min. Cells were washed and resuspended sequentially in three lysis buffers (Buffer 1: 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100; Buffer 2: 10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; Buffer 3: 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) to isolate chromatin. Chromatin was sonicated for 15 cycles (20s on, 30s off, 25% amplitude) using an EpiShear probe sonicator (Active Motif). Sonicated chromatin was incubated overnight at 4°C with 3µg of antibody for histones or 10µg of antibody for other proteins. Next, 50µl of Protein G magnetic beads (Invitrogen, 10004D) was added and incubated for 4h at 4°C. Magnetic beads were washed and the chromatin eluted, followed by de-crosslinking and DNA purification. The ChIP and input DNAs were analyzed by quantitative PCR (qPCR) using intergenic regions as negative controls (chr2:73,030,265-73,030,373; chr6: 52,339,345-52,339,505). The qPCRs for each sample were performed as technical triplicates.