Graphical Abstract Highlights ChIP-seq is a prevailing methodology to investigate and compare epigenomic states Lack of an empirical normalization strategy has limited the usefulness of ChIP-seq ChIP-Rx allows genome-wide quantitative comparisons of histone modification status ChIP-Rx identifies graded epigenomic changes following chemical perturbations SUMMARY Epigenomic profiling by chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is a prevailing

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... ogy used to investigate chromatin-based regulation in biological systems such as human disease, but the lack of an empirical methodology to enable normalization among experiments has limited the precision and usefulness of this technique. Here, we describe a method called ChIP with reference exogenous genome (ChIP-Rx) that allows one to perform genome-wide quantitative comparisons of histone modification status across cell populations using defined quantities of a reference epigenome. ChIP-Rx enables the discovery and quantification of dynamic epigenomic profiles across mammalian cells that would otherwise remain hidden using traditional normalization methods. We demonstrate the utility of this method for measuring epigenomic changes following chemical perturbations and show how reference normalization of ChIP-seq experiments enables the discovery of disease-relevant changes in histone modification occupancy.