Improved spike-in normalization clarifies the relationship between active histone modifications and transcription
Spike-in normalization enables quantitative analysis of ChIP-sequencing (ChIP-seq) signal. Here we introduce a novel robust dual-spike-in normalization approach for ChIP-seq (ChIP-wrangler) and revisit recent claims that active histone marks are dependent on transcription. Concerned that previous studies improperly used spike-in normalization to arrive at their conclusions, we used ChIP-wrangler to show that acute depletion of RNA polymerase II has only a modest impact on the levels of H3K4me3 and H3K27ac. In line with other studies, our results provide proof that the maintenance of histone acetylation is not merely a consequence of ongoing transcription. Our innovative ChIP-seq normalization approach provides increased rigor and “guardrails” for successful spike-in normalization, and as applied here refines the understanding of the intricate crosstalk between RNA polymerase II activity and histone marks associated with transcription Overall design: HCT116-EF1a-OsTIR1-Rpb1 cells were treated with either 500nM Triptolide (Sigma-Aldrich T3652) or 500uM auxin (3-indoleacetic acid, Sigma-Aldrich #I3750) to degrade Rpb1 for 0, 1, and 4 hours. HeLa-S3 cells were treated with 500nM Triptolide (Sigma-Aldrich T3652) for 0, 1, or 4 hours. Sheared chromatin from D. melanogaster and S. cerevisiae were added as spike-in controls to all samples prior to immunoprecipitation. From every biological sample, a ChIP-input sample was prepared to assess the relative amounts of spike-in chromatin. ChIP-seq was perfomed using antibodies for H3K27ac, H3K4me3, H3K4me1, H3K36me3, and Rpb1 in HCT116-EF1a-OsTIR1-Rpb1 cells, and antibodies for H3K27ac, H3K4me3, H3K4me1, and Rpb1 in HeLa-S3 cells.