GSE212386
Analysis of histone antibody specificity directly in sequencing data using siQ-ChIP
We previously developed sans spike-in quantitative chromatin immunoprecipitation sequencing (siQ-ChIP), a technique that introduces an absolute quantitative scale to ChIP-seq data. The absolute scale is possible because the IP step of ChIP is a competitive binding reaction and produces a classical binding isotherm when antibody or epitope is titrated. The mathematical model that led to siQ-ChIP also predicts that sequencing samples from along an isotherm can reveal differential preferences of the antibody for specific chromatin regions, if the antibody binds with different strengths to different regions. In this paper, we directly test this prediction with several histone post-translational modification (PTM) antibodies. When titrating these antibodies, we identified on- and off-target interactions directly in ChIP-seq. Antibodies that displayed mostly on-target interactions had small, uniform responses for different regions of chromatin, while antibodies that contained off-target interactions had large, variable responses, indicating differences in binding affinity for different chromatin regions. The generation of an isotherm is essential to perform siQ-ChIP analysis. Therefore, we also present a detailed and simplified wet lab protocol, optimized to reproducibly achieve a binding isotherm. We highlight several benchmarks of success that can be used to gain confidence in ChIP experiments, serving as a guide for the practice of siQ-ChIP. Collectively, these studies demonstrate an optimized siQ-ChIP protocol that can be used to determine histone PTM antibody specificity directly in sample chromatin without any exogenous spike-in reagents and at minimal sequencing depth. Overall design: HeLa ChIP-Seq