Timing RNA polymerase pausing with TV-PRO-seq

Various next-generation sequencing-based methods, including Pol II ChIP-seq, GRO-seq, NET-seq, and PRO-seq, have been able to reveal the presence of polymerase pausing. However, these methods are mainly based on detection of polymerase occupancy, which is affected by factors other than the pausing. As an example, recent research revealed that polymerase has a high abortive transcription rate, especially at genes featuring strong pausing, which also contributes to high polymerase occupancy in the promoter-proximal regions. Further, the accuracy of the most widely used method to measure pausing—treating cells with triptolide (Trp)—is challenged by the latter's slow uptake. A method that outputs the actual pausing time is still required to dissect the pausing profile. We have developed TV-PRO-seq, which reveals polymerase pausing times at single-base resolution genome-wide and is not influenced by the polymerase turnover and other confounding factors