Dissecting polymerase pausing with TV-PRO-seq

Transcription of many genes in metazoans is subject to polymerase pausing, which corresponds to the transient arrest of transcriptionally engaged polymerase. It occurs mainly at promoter proximal regions and is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking. This is because relevant experimental techniques are mostly based on average polymerase occupancies across many cells which cannot discriminate between few slow polymerases and many quick polymerases at a position, since the data corresponds to the average of many cells. We present here an extension of the PRO-seq assay, which we termed time variant PRO-seq (TV-PRO-seq)based on a detailed analysis of the logics underlying PRO-seq, that achieves this goal. Overall design: The main TV-PRO-seq experiment consisted of 8 independent PRO-seq samples : biological duplicates of the 4 run-on times 30sec, 2min, 8min and 32min. The TV-PRO-seq have been performed in 2 human cell lines KBM7 and HEK293. Two triptolide treatment sample have been taken in KBM7 cells for 3min and 6min run-on, DMSO treatment have been use for control for the same run-o time.