ChIP Assay Protocol for Protein-DNA Interactions

ChIP assay was performed as described previously ^{8 (link)}. 2.5 X 10^{6 (link)} cells were taken for ChIP assay. The whole cell extract was pre–cleared using 60 μl of Protein A/G PLUS – agarose beads (SCBT; sc–2003) and incubated overnight with anti–P53, anti–H3K4me3, anti–Wdr5, anti–RbBP5, anti–Ash2L or control IgG antibodies. 1 μg of antibody was used for 100 μg of total lysate. Protein G agarose beads pre–blocked with salmon sperm DNA (to reduce non–specific DNA binding) was purchased from Millipore, Saint Charles, MO, USA (#16–2001) and 60 μl of this solution was used for each immunoprecipitation. 10 % of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (input). Both Immunoprecipitated and whole cell extract (input) were treated with RNaseA, proteinase K and DNA were purified using DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA; #69504). Quantitative real–time PCR was performed on this DNA to identify the amount of target sequence. The list of primers used is given in supplementary table.