Fig. 4: Pol II entering the gene body in the absence of Cdk9 is largely restricted to the first 3-4 kilobases.

a A schematic of a Flavopiridol time-course that was performed on Drosophila S2 cells in the presence or absence of NELF knockdown (as in Fig. 1). Spike-in normalized PRO-seq was again used to accurately quantify the effects on active transcription. b Illustration of hypothetical PRO-seq profiles (not to scale). In both NELF knockdown and the LacZ control, we predicted that a “clearing wave” of gene body Pol II would be observed over the time-course, but that the NELF knockdown would also permit a slower “advancing wave” similar to what has been observed for the fission yeast S. pombe, which lack NELF. c, d PRO-seq (RRPM/kb calculated within 200 bp bins) at representative genes. The long gene cv-c c demonstrates the full extent of clearing waves, while the shorter gene Lrch d is entirely cleared during the time-course. Double lines indicate signal exceeding the y-axis limit. Arrows provide matching points of reference. e, f For each RNAi condition, metagene profiles of PRO-seq log fold-changes (within 1 kb bins, bootstrapped mean + 50% confidence interval) following FP are plotted for filtered genes >55 kb (n = 39). g PRO-seq metagene profiles (bootstrapped mean + 50% CI within 100 bp bins) for filtered genes >6.8 kb in length (n = 469). h For each drug treatment, metagene profiles (bootstrapped mean + 50% CI) of PRO-seq log fold-changes in NELF vs. LacZ RNAi are calculated in 200 bp bins for each filtered gene >16 kb (n = 227). Arrows mark TSS + 3.6 kb, the average extent of the aberrant density after 20’ FP.