ChIP-qPCR Protocol for Chromatin Immunoprecipitation

ChIP experiments were performed from 30–50 μg MCF-7 chromatin essentially as previously described (Chapman et al., 2013 (link)). Briefly, chromatin was immunoprecipitated using 3 μg anti-p53 (DO-1, Santa Cruz), 1.5 μg anti-RNAP2 CTD (phospho-S5; Clone 4H8), 2.5μg Anti-Histone H3 (acetyl K9) (ab4441, Abcam), or 2.5 μg anti-acetyl-histone H4 (06-598, Millipore) antibody coupled to 25 μl Protein-A/G Dynabeads (Life Technologies). DNA quantities recovered in control IgG ChIP experiments were consistently below the detectable range. Relative quantities of ChIP-enriched DNA were calculated relative to total input chromatin by qPCR in triplicate on a CFX96 Real-Time Analyzer (Bio-Rad) using Quantifast SYBR Green reagent (QIAGEN) and locus-specific primer pairs (sequences in Supplemental Experimental Procedures).