Fig. 2: EM-based visualization and quantification of R-loops on human genomic DNA upon estrogen-dependent transcriptional burst.
From: Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids
a, qPCR–DRIP analysis of MCF7 cells with or without 2 hours of E2 stimulation and with or without in vitro RNase H digestion (H) for representative E2-inducible and constitutive genomic loci. Data were normalized to a negative (neg.) locus and is shown as mean ± s.d., n = 3 independent biological replicates. Statistical significance was determined by unpaired two-tailed t-test. Inducible and constitutive loci differ significantly, as determined by two-way ANOVA (P = 5.78 × 10–7). b, Dot blot analysis of genomic DNA extracted from MCF7 cells with or without 2 hours of E2 stimulation and with or without in vitro RNase H digestion. Genome-wide hybrid accumulation was detected by S9.6 immunostaining (loading control: dsDNA). c, Quantification of integrated intensities in b; S9.6 signal was normalized to the respective dsDNA loading control. a.u., arbitrary units. d–g, EM analysis of R-loops on genomic DNA extracted from MCF7 cells with or without 2 hours of E2 stimulation and with or without in vitro RNase H digestion. This analysis was performed in two independent biological replicates (additional data in Extended Data Fig. 3). d, Representative electron micrographs of R-loops (indicated by the black arrows) found on gDNA from E2 stimulated cells. Scale bars, 200 bp/72 nm. e, Sizes of single R-loops in bp. Black lines and gray numbers indicate the median R-loop size. f, Frequency of R-loops that are >300 bp in size. Absolute numbers of R-loops were normalized to the total DNA content within the analyzed area. g, Total R-loop burden, calculated as the genomic fraction of bp involved in R-loop formation.
