Extended Data Fig. 2: Optimization of EM sample preparation to detect R-loop structures on genomic DNA.
From: Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids
a–b) qPCR-DRIP analysis of MCF7 cells +/− 2 h E2 +/− in vitro RNase H digestion for representative E2-inducible and constitutive genomic loci on the extracted genomic DNA used for the two biological replicates of the EM analysis. Data was normalized to a negative locus. a) corresponds to data shown in Fig. 2b-g, b) corresponds to data shown in Fig. 2e–g, Extended Fig. 2c, d and Extended Data Fig. 3a, b. c) Dot blot analysis of genomic DNA extracted from MCF7 +/− 2 h E2 +/− in vitro RNase H digestion. Genome wide hybrid accumulation was detected by S9.6 immunostaining (loading control: dsDNA). d) Quantification of integrated intensities in b); S9.6 signal was normalized to the respective dsDNA loading control. e) Genomic DNA, extracted from E2 stimulated MCF7, was digested with different cocktails of restriction enzymes: 1. PvuII, 2. PvuII + EcoRI, 3. NotI, 4. BbvCI, 5. HindIII + EcoRI + BsrGI + XbaI + SspI. Hybrid levels were detected by S9.6 dot blot (S9.6 blot shown as short and long exposure; loading control: dsDNA). Digestion cocktail 2 was used for all follow-up experiments. This result was reproduced once. f) Agarose gel electrophoresis of samples in e) showing the different degrees of genomic DNA fragmentation. g) Dot blot of extracted genomic DNA, extracted from E2 stimulated MCF7 and purified either directly or after restriction enzyme digest using either size exclusion columns (Amicon) or a silica bead extraction kit (SBK). Note that, for unknown reasons, hybrids are not recovered from Amicon columns, which is why SBK was selected for all follow-up experiments. Heat inactivation was used as positive control for hybrid stability. Hybrid stability was assessed by S9.6 dot blot (S9.6 blot shown as short and long exposure; loading control: dsDNA). This result was reproduced once. h) Agarose gel electrophoresis of samples in g) showing the degree of genomic DNA fragmentation. i) Automated high-throughput EM workflow used to image and quantify R-loops on human genomic DNA43.
