Additional file 1: of DNA Topoisomerase I differentially modulates R-loops across the human genome
Creators: name: Manzo, Stefano
name: Hartono, Stella
name: Sanz, Lionel
name: Marinello, Jessica
name: Biasi, Sara De
name: Cossarizza, Andrea
name: Capranico, Giovanni
name: Chedin, Frederic
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Figure S1. A. Quantitation of R loop signal from dot blot shown in Fig. 1. Error bars are SE of three independent experiments. A representative DNA loading control is shown at right after agarose gel electrophoresis. B. Dot blot and relative quantitation to measure R loop loads in genomic DNA from control or Top1 depleted cells, with and without RNase H pre-treatment. C. Dot blot depicting change of global R-loop signal 4, 5, and 6 days after initial Top1 depletion. Samples are indicated at left. D. Distribution of DRIPc signal (right) across genomic compartments compared to expected distribution in the genome (left). Color codes are as described in Fig. 1c. E. Genome browser screenshot illustrating signal “spreading” in the Top1-depleted samples. The R-loop signal for the (+) strand is shown in red. The high-sensitivity peak calls developed to capture signal spreading are found below each track. F. XY plot of average log(10) count-normalized DRIPc-seq signals for control samples (scramble – x axis) and top1-depleted samples (y-axis). G. Validation by DRIP-qPCR of R-loop gain and loss loci identified by DRIPc-seq. Error bars are SE of three independent experiments. H. Validation by DRIP-qPCR R-loop gain (left) and loss (right) upon depletion of Top1 by a second, independent siRNA. The inset above shows a Western blot verifying Top1 depletion. I. Validation by DRIPqPCR of RLL and RLG loci identified by DRIPc-seq 5 and 6 days after Top1 depletion. Error bars represent SE of 2 independent experiments. J. Validation of R-loop loss and gain loci identified by DRIPc-seq using S9.6-independent DRIVE-pPCR method. The average and standard deviation of two independent replicates is shown. The RPL13A locus represents an invariant control. K. DRIPqPCR analysis of R-loop formation over the 5’ ETS and 28S rDNA regions. Results are average of 3 independent experiments shown with standard deviation. RNase A and RNase H pre-treatments are indicated below. L. DRIPqPCR analysis of R-loop formation over the 5’ ETS and 28S rDNA regions with a second siRNA against Top1. Results are average of 2 independent experiments shown with SEM. Figure S2. Examples of DRIPc profiles for control and Top1-depleted cells for genes showing gain and loss (A) or mixed R-loop changes (B) after Top1 knockdown. Stars indicate statistically significant differences. C. Ratio plots of the RNA polymerase II ChIP-seq signal between Top1-depleted and control cells around the TSS of specific gene categories according to expression and pausing status. Top1 depletion causes an increase in RNAPII levels around the TSS of paused genes. Peak shape differences between our and previous studies 30 are likely due to the use of different antibodies (we used a pan-RNAPII Ab whereas others used an anti-S5P RNAPII Ab). This allowed us to observe progressive RNAPII accumulation downstream of the TSS, consistent with RNAPII encountering difficulty during elongation in the absence of Top1. D. Venn diagrams depicting the overlap between RLG and RLL genes and genes undergoing up or down regulation in Top1-depleted cells. Differentially expressed genes were identified with a 1.5-fold up or down minimal threshold (and adjusted p-value 50%; no star