R-loops promote antisense transcription across the mammalian genome

We report the correlation we found between R-loops and antisense trasncription initiation. Our in vitro data suggests that RNA pol II can initiate transcription off the displaced single stranded DNA from within the R-loop. Therefore we generated Chromatin associated RNA-seq with and without over-expressing RNaseH1 and RDIP-seq (the directional RNA-seq of the RNA moeity from within the S9.6 pull down R-loop) as well as RR-ChIP-seq (the directional RNA-seq of the RNA moeity from within the catalytically dead RNaseH1-GFP GFP pull down R-loop) and confirm the correlation in vivo. We also aligned our findings with ChrCAP-seq (which is is a library enriched for RNA polymerase II capped RNA using chromatin associated RNA as the initial source) and found that capped antisense RNA were sensitive to RNase H1 overexpression. Overall design: Examination of chromatin associated RNA of HeLa cells and capped RNA with and without over-expressing RNAseH1. Directional R-loop occupancy was conducted by 1) sequencing the RNA moeity of the R-loop fragments which were immunoprecipitated by mouse monoclonal S9.6 antibody and by 2) sequencing the RNA moeity of the RNA:DNA hybrids caught in a catalytically dead RNaseH1-GFP.