R-loops are increased at intergenic SETX-binding sites. (A) Genome browser views of R-ChIP and input control signal in wild-type and SETX knock-out U2OS cells at representative genome locations. Two biological replicates were used and ChIP signal for each line is shown after the removal of input contribution as well as read depth normalization. (B) Normalized R-ChIP signal from wild-type and SETX knock-out cells at promoter sites (TSS) with standard error. TSS locations determined from the dominant transcript for each protein-coding gene (highest number of reads) using poly(A)-selected RNAseq data from wild-type U2OS cells. N = 13,341. (C) Visual representation of gene body, intergenic, or promoter locations of R-ChIP peaks called by MACS2 call peak for wild-type and SETX knock-out R-ChIP as well as SETX ChIP (Cohen et al., 2018). (D) Genome browser views of R-ChIP and input control signal in wild-type and SETX knock-out U2OS cells at representative intergenic genome locations. (E) Visualization of SETX KO R-ChIP signal at locations of SETX binding (called peaks from SETX ChIP no DSB treatment SETX_mOHT from Cohen et al. [2018]); “center” is center of SETX ChIP peak. (F) Quantification of R-ChIP signal from wild-type and SETX KO U2OS cells at locations of SETX binding in the absence of DNA damage; graph showed input-normalized, log₂ transformed ChIP signal. *** indicates P < 0.0001 by two-tailed Mann–Whitney test. (G) Accumulated SETX ChIP signal at locations within genes (left panel) and intergenic R-ChIP peak locations (right panel) in SETX knock-out cells.