Antibody background in ChIP-seq skews estimates of cohesin positioning by CTCF barriers

Abstract

Loop extruding cohesin complexes are positioned by CTCF barriers to generate locus-specific 3D genome folding patterns. Quantifying cohesin accumulation at CTCF sites is crucial for deriving insight into loop extrusion and its consequences. Here we confronted expectations from loop extrusion simulations with experimental data by developing a pipeline, ChIP-FRiP, and analysis framework to reliably quantify cohesin positioning. We used ChIP-FRiP to uniformly re-process 140 cohesin ChIP-seq datasets from 13 publicly available studies. This analysis revealed that non-specific antibody binding can skew measurements of cohesin positioning. To mitigate this bias, we developed a biochemical model of background ChIP-seq signal and a strategy to estimate and correct this background using spike-in ChIP-seq data and relative protein abundance before and after depletion. Our results establish a framework for comparative analysis, demonstrating that accurate background correction is requisite for interpreting the roles of cohesin cofactors in cohesin positioning.