(A) DNA-RNA hybrids were quantified in undamaged U2OS cells by monitoring chromatin-bound RNaseHD10R-E48R-mCherry by FACS. 10,000 cells were counted in each of 3 biological replicates; error bars represent S.D. * and ** denote p < 0.05 or 0.01, respectively in Student's two-tailed T test with comparisons as indicated. (B) RNA/DNA hybrids were quantified in undamaged, CtIP-depleted U2OS cells complemented with either vector only, eGFP-CtIP(wt), or nuclease-deficient eGFP-CtIP(NA/HA) as in (A). 10,000 cells were counted in each of 3 biological replicates; error bars represent S. D. **** denotes p < 0.0001 using Student's two-tailed T test with comparisons as indicated. (C) S9.6 antibody was used to monitor RNA-DNA hybrids in wild-type or CtIP-depleted U2OS cells. Anti-Nucleolin was used as a marker for the nucleolus. (D) Quantification of S9.6 antibody signal in wild-type, CtIP-depleted, XPG-depleted, Senataxin-depleted, or double CtIP/XPG-depleted U2OS cells as indicated. Signal overlapping the nucleolin signal was excluded from the analysis; n > 50, Error bars represent S.E.M. *, **, and **** denote p < 0.05, 0.01, and 0.0001, respectively, using Student's two-tailed T test with comparisons as indicated. (E) Quantification of S9.6 antibody signal in wild-type, CtIP-depleted, or CtIP-depleted plus RNaseH overexpression in U2OS cells as in (D). (F) Wild-type or CtIP-depleted U2OS cells were exposed to CPT (5 µM) or were untreated before harvesting of cellular mRNA. Analysis of transcripts by RNA-seq and hierarchical clustering of transcripts from 21,412 genes is shown as a heat map (red for over-expressed, black for unchanged expression, and green for under-expressed genes) in comparison to wild-type undamaged cells (see Supplementary file 3). (G) Statistical comparisons of overlap between the top 100 differentially expressed genes as ranked by DESeq differential expression p-value and DRIPc-seq peaks from GEO dataset GSE70189. Randomly picked genes were compared to this dataset (‘randomized control’) with the average of 35.56% and standard deviation 4.76% (estimated from 1000 simulations). Genes with significant differences between wild-type and CtIP-depleted cells were identified in the absence of DNA damage (‘WT vs CtIP shRNA (-CPT’)) as well as with CPT treatment (‘WT vs CtIP shRNA (+CPT’)) and were compared with the DRIPc-seq dataset. All three values are above the 99% confidence intervals for the null hypothesis that the genes showing the most evidence of differential expression overlapped the peak regions at the same rate as randomly selected genes.