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