Extended Data Fig. 1: Direct visualization of in vitro-generated R-loops using Electron Microscopy (EM).
From: Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids
a) pFC53 map including the R-loop prone mAIRN gene, the T3 promotor used in this study and the XmnI linearization site. b) Gel shift assay of circular and linear pFC53 +/− RNase H treatment. This result has been reproduced 3 independent times. c) Dot blot of circular and linear pFC53 +/− RNase H, immunoblotted for S9.6 and dsDNA (as loading control). When indicated, pFC53 was incubated with 20% formamide and 0.02% BAC for 1 min prior to dot blot loading. This result has been reproduced 3 independent times. d-f) Competition experiment to assess the specificity and selectivity of the S9.6-gold conjugated antibody. The linearized competition plasmid and R-loop carrying pFC53 were mixed in the indicated ratios, labeled with S9.6-gold and spread for EM analysis. For each plasmid at least 100 molecules were analyzed. The experiment was reproduced once. d) Length distribution of the imaged fragments. pFC53 and the competition plasmid were identified by their respective sizes. The relative frequencies of pFC53 and competition plasmid displayed do not represent the frequencies observed in the sample. e) Quantification of S9.6-gold binding to the pFC53 and the competition plasmid. Binding to pFC53 was further differentiated into specific (within the mAIRN gene) or unspecific (outside of the mAIRN gene). f) Calculated R-loop density (number of R-loops/Mb) for the ratios indicated below.
