Costantino L & Koshland D The Yin and Yang of R-loop biology. frequencies.A. The graph shows the log2 fold ratio of observed (R-loop forming) over BKM120 (NVP-BKM120, Buparlisib) expected (matched non-R-loop forming) frequencies for each 6-mer. Some 6-mers are clearly more or less represented than others in DRIPc-seq data compared to expectations from control non-R-loop loci. This could reflect the intrinsic sequence preference of R-loop formation and/or the intrinsic preference of S9.6 antibody. If the latter is true, we expected S9.6-highly bound epitopes (red) to be over-represented and S9.6-poorly bound epitopes (blue) to be underrepresented. This was not observed, however. Instead, S9.6 tightly or poorly bound 6-mers were equally likely to be under- or over-represented. This suggests that DRIPc-seq data does not suffer from systematic biases caused by S9.6 sequence preference. B. To account for what could be driving the over- or under-representation of certain 6-mers, we simply calculated the GA content of the motifs. As shown below, depleted motifs tend to be GA-poor (CT-rich), while enriched motifs tend to be GA-rich irrespective of whether they are tightly or poorly bound by S9.6 (the dashed grey line represents 50% GA content). Given that GA-rich regions are favorable for R-loop formation, the observed trends are most likely to reflect the intrinsic sequence biases underlying R-loop formation, not S9.6 binding. Similar results were observed when 8-mers were considered. Supplementary Figure 2: Genomic DNA digestion profiles. DNA digestion profiles after Step 10 were visualized after agarose gel electrophoresis through a 0.8% agarose gel run in 1x TAE buffer. DNA was extracted from human NTERA-2 cells and digested with restriction enzyme cocktail indicated in Step 10. Lanes 1 and 2 show an example of incomplete digestion, as evidenced by the high molecular weight bands above 20 kilobases. Lanes 3 and 4 show an example of AURKB fully digested DNA as judged from the disappearance of the top band. The leftmost lane (M) corresponds to a 1kb plus GeneRuler ladder from ThermoFisher. NIHMS1036071-supplement-1.pdf (4.9M) GUID:?85372177-0E94-4F81-A7EE-72D70FEECF9A Abstract R-loops are prevalent three-stranded non-B DNA structures composed of an RNA:DNA hybrid and a single-strand of DNA. R-loops are implicated in various basic nuclear processes such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest over recent years. Here, we describe two related immunoprecipitation-based methods to map BKM120 (NVP-BKM120, Buparlisib) R-loop structures: basic DRIP-seq (DNA:RNA ImmunoPrecipitation followed by sequencing), an easy, robust, but resolution-limited technique, as well as DRIPc-seq (DNA:RNA Immunoprecipitation followed by cDNA conversion and sequencing), a high-resolution and strand-specific iteration of the method that BKM120 (NVP-BKM120, Buparlisib) permits accurate R-loop mapping genome-wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA:DNA hybrid S9.6 antibody. Compared to DRIP-seq in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops and these RNA strands are subjected to strand-specific RNA-seq analysis. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles, and dynamic resolution of these important structures. Keywords: R-loops, DNA:RNA hybrids, S9.6, DRIP, DRIPc, library, mapping, strand-specific RNA-sequencing EDITORIAL SUMMARY: R-loops are DNA:RNA hybrid structures found throughout the genome and relevant to both normal and disease states. DRIPc-seq, which is based on immunoprecipitation with the S9.6 antibody recognizing DNA:RNA hybrids, permits genome-wide mapping of R-loops. TWEET: DRIPc-seq accurately maps genome-wide BKM120 (NVP-BKM120, Buparlisib) distribution of R-loops following immunoprecipitation with an anti-DNA:RNA hybrid antibody. COVER TEASER: Genome-wide mapping of R-loops with DRIPc-seq Introduction R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and a single-stranded DNA loop. These structures form primarily during.