Fig. 3: Antagonistic genomic distribution of NDX binding sites and R-loops.

a Genome browser snapshot showing the enrichment of flag-NDX and DRIP signals along chromosome 3. RNase H-treated sample represents the background DRIP signal. RNA-DNA hybrids were immunoprecipitated by S9.6 monoclonal antibodies. b Venn diagram showing the overlap of flag-NDX peaks and DRIP peaks (R-loops). The number of common peaks is not equal because ChIP peaks can overlap with multiple DRIP peaks and vice versa. c Pile up plot showing the antagonistic distribution of ChIP and DRIP signal intensities over the predicted peak positions. RNase H treatment is shown as a negative control for DRIP. Color scale corresponds to RPGC (reads per genomic content) values. Peak summits were aligned to zero positions. d Metagene profile of flag-NDX (light blue), NDX-GFP (light green) and DRIP signal (red) intensities (mean values) over protein coding ORFs. TSS: transcription start site. TTS: transcription termination site. e DRIP-qPCR validation of representative DRIP peaks. Specific DRIP signal and background (+RNase H) signal are shown in red and black, respectively. Genomic positions and qPCR amplicons are indicated. Error bars represent SEM. f Secondary ssDNA structure prediction over NDX binding sites and RNA-DNA hybrids. The plot shows the propensity of single-strand formation of individual flag-NDX peaks and DRIP peaks, common NDX/DRIP peaks, and randomly selected regions. Sample size n = 300 peaks randomly selected from DRIP and ChIP peak lists, and n = 300 random peaks. The ssCount values were calculated from the primary nucleic acid sequence of the peaks using the mfold algorithm. Statistical significance and p values are indicated (Mann-Whitney U test, two-sided). Bounds of boxes describe the interquartile range with the median; whiskers indicate minimum and maximum values; outliers are not shown.