Theoretical and practical refinements of sans spike-in quantitative ChIP-seq with application to p300/CBP inhibition

Abstract

Previously, we introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing. The scale itself was determined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. In this paper we extend those results in several ways. First, we simplified the calculations defining the quantitative scale. Second, we highlight the normalization constraint implied by the quantitative scale and introduce a new scheme for generating tracks for siQ-ChIP. We next introduce some whole-genome analyses that are unique to siQ-ChIP which allow us, for example, to project the IP mass onto the genome to evaluate how much of any genomic interval was captured in the IP. We apply these analyses to p300/CBP inhibition and demonstrate that response to inhibition is a function of genomic architecture. In particular, active transcription start sites are only weakly perturbed by p300/CBP inhibition while enhancers are strongly perturbed. Similar observations have been reported in the literature, but without a quantitative scale, those observations have been misinterpreted. We discuss how the siQ-ChIP approach precludes such misinterpretations, which stem from the widespread community practice of treating unquantified and unnormalized ChIP-seq tracks as though they are quantitative.