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 detemined 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.

Analysis of histone antibody specificity directly in sequencing data using siQ-ChIP

“…Using the protocol described herein, one can achieve a reproducible binding isotherm for a given antibody without spike-ins and obtain an absolute scale for sequencing quantification. We have gone on to use several different antibodies in ChIP-seq in this paper and others 10 and have yet to fail in generating a binding isotherm, unless the antibody did not bind to magnetic beads, as was the case for Ab7 mentioned above. Sensitivity of the chromatin-antibody isotherm is essential for concordance with the competitive binding model.…”

“…4D ). The conceptual and mathematical derivation of these analyses is described in our prior work 10 .…”

“…To compare responses between titration points for each antibody from Figure 4 , we next analyzed peak areas of siQ-ChIP normalized tracks 10 . Recalling from earlier, the y-axis is the siQ-ChIP capture efficiency, which is normalized so 1 represents 100% of IP capture of input.…”

“…We previously developed a physical and quantitative scale with which to interpret ChIP-seq outcomes 3, 10 . This method, sans spike-in quantitative ChIP-seq (siQ-ChIP), is an inherently quantitative approach that does not require spike-ins for signal normalization.…”

Ketolysis is a metabolic driver of CD8+ T cell effector function through histone acetylation

“…ChIP-seq analysis of H3K27Ac enrichment in CD8 + T cells was conducted using the sans spike-in quantitative ChIP (siQ-ChIP) method as previously described ( 24, 25 ). Naïve CD8 + T cells (5 x 10 6 ) were activated with anti-CD3 and anti-CD28 antibodies for 24 h, then rinsed once with 10 mL of D-PBS (Gibco, 14190136) followed by cross-linking in suspension for 5 min in 10 mL of 0.75% formaldehyde (Pierce, 28906) in D-PBS at room temperature.…”