Genome wide mapping of R-loops in the repriming deficient human cells using RNA DIP-Seq

The goal of this study was to compare the qualitative and quantitative differences in the presence of DNA:RNA hybrids (R-loops) between human wild type and PrimPol knockout human iPS cell lines. Our initial genetic experiments on a reporter locus have showed a link between the formation of R-loop and non-B DNA motifs; we have therefore analysed the correlation between R-loop abundance and of sequences computationaly predicted to form triplex-DNA or G-quadruplex forming sequences, and whether this changes between the two cell lines. We examined abundance of R-loops in wild type and PrimPol deficient BOBSC iPS cell lines using an immunoprecipitation approach using DNA:RNA hybrid specific monoclonal antibody S9.6. To allow for quantitative comparisons between the cell lines, prior to extraction we have spiked-in each mutant cells with the same ratio of chicken DT40 cells to act as a calibrator.