R-loop Mapping with DRIP Protocol Modifications

R-loop mapping was performed following the DRIP protocol^{50 (link)} with some modifications. PATC53 cells were treated with 1 µM PRMTi or DMSO as control. One and three days later, DNA was extracted as described^{76 (link)}, but genome fragmentation was conducted by shearing via sonication using a Diagenode Bioruptor (12 cycles, High, 15’ON 90’OFF). As sonication degrades the single-stranded looped out DNA strand of R-loops, immunoprecipitation with S9.6 enriches mostly for two-stranded RNA: DNA hybrids. To build sequencing libraries, hybrids were transformed back into double-stranded DNA via a second-strand DNA synthesis step using E. coli RNase H1, DNA ligase, DNA polymerase I (New England Biolabs) and a dNTP mix in which dUTP was used instead of dTTP. After checking the quality of the immunoprecipitation by qPCR, the DNA was built into strand-specific sequencing libraries with a UDG DNA glycosylase step before the PCR amplification step to ensure strand specificity^{76 (link)}. Library quality was checked on an Agilent BioAnalyzer and sequencing performed on an Illumina HiSeq4000 instrument. Mapping was performed on two independent biological replicates.