Fig. 1: HiS-NET-seq provides a more complete view on transcriptionally engaged RNA polymerase.

a Schematic view of the main steps of the HiS-NET-seq approach. The sequencing primer is indicated as black arrow (right panel). 4sU: 4-thiouridine (4sU, red); unique molecular identifier (UMI, blue). b Pearson’s correlation analysis of Pol II occupancy per active gene for two biological replicate measurements of HiS-NET-seq (r = 0.99). Human gene counts were RLE-normalized (see Methods), and 0.5 pseudo counts were added. c Barplot shows the fraction of sequencing reads that mapped to Pol II transcribed regions (described in Methods) and sn/snoRNA genes. Unmapped sequencing reads and reads of masked regions are not shown. Fractions are indicated for HiS-NET-seq (top) and NET-seq (lower panel) data. d RPM normalized Pol II occupancy for individual nucleotides at indicated regions. Excluded were signal outliers above the 99.99- or 99.9-quantile in HiS-NET-seq or NET-seq data, respectively. Pol II density at transcription start sites (TSSs) and polyadenylation (pA) sites was masked. TSSs and transcription directions are indicated by black arrows. Enhancers without Pol II signal in either HiS-NET-seq or NET-seq are not shown. kb: kilobase. e Quantification of Pol II occupancy measured by HiS-NET-seq and NET-seq, respectively. Element types as described in the Methods section include active genes (n = 9454) and FANTOM5 enhancers (n = 6313). Percent of transcribed elements with indicated TPM threshold or higher. f Pol II occupancy for individual nucleotides at gene regions (n = 11,303). Excluded were signal outliers above the 99.99-quantile in all datasets. The region from the TSS + 1.5 kb to the polyA site was scaled to 0.5 kb (gray). b–d, f Mean HiS-NET-seq values from two biological replicate measurements are shown. b–f Data were obtained for human K562 cells.