Supplementary Figure 4: Validation of nascent-RNA sequencing.

(a) Heat maps of nascent RNA-seq are displayed for Topo I- (left) and Topo II-inhibited (right) samples as performed in Fig. 4b but with genes arranged by increasing gene length. The 3' end of long genes experiences a decrease in nascent RNA after Topo I inhibition. (b) Heat shocked samples were processed for nascent RNA and sequencing in parallel with untreated control and topoisomerase inhibited samples. The log ratio of heat shocked over control was calculated and displayed as heat map for regions 2 kb surrounding the TSS and TES with all genes arranged in decreasing expression of control samples (top). As expected, the nascent RNA levels decreased for most transcribed genes following 15 minutes of heat shock. Also as expected, the highly induced Hsp genes showed increased expression (bottom). (c) Based on the sequencing results, 3 groups of genes were selected: down-regulated, no change, and up-regulated after Topo I inhibition. Primers were designed for each of the genes and were used to perform qPCR analysis on replicate nascent RNA preparation. qPCR results show similar trends with sequencing data.