Q3. How have R-loops been mapped on a genome-wide scale?
- DRIP-seq, the first method for genome-wide mapping of R-loops, was initially developed by Fredric Chedin group in 2012 (PMID: 22387027). Basically, isolated genomic DNA is fragmented via a combination of restriction enzymes. R-loop-containing fragments are then enriched with S9.6 antibody and subjected to sonication, dsDNA end repair, adaptor ligation and PCR amplification for deep sequencing.
- DRIVE-seq was also developed by Fredric Chedin group at the same time of DRIP-seq (PMID: 22387027). The key difference with DRIP-seq is that human RNase H1 mutant instead of S9.6 is adopted for in vitro enrichment of RNA:DNA hybrids.
- RDIP-seq, another variant of DRIP-seq-like technology (PMID: 26579211), uses RNase I to remove any single-strand RNA from the sample, and sonication to minimize sequence bias by restriction digestion. Furthermore, template DNA is used for RNA-primed extension with dUTP and strand-specific library construction and sequencing.
- S1-DRIP-seq, developed by Douglas Koshland group in 2016 (PMID: 27298336). The method is identical to DRIP-seq except that genomic DNA is treated with S1 nuclease prior to sonication to stabilize R-loops, then immunoprecipitated with S9.6 antibody and sequenced.
- DRIPc-seq, as a modified version of DRIP-seq, was developed by Fredric Chedin group in 2016 (PMID: 27373332). Instead of sequencing dsDNA, the RNA moiety of R-loop structures is sequenced in a strand-specific manner.
- ssDRIP-seq, developed by Qianwen Sun group in 2017 (PMID: 28848233), uses more freqent cutters for DNA fragmentation. With further sonication, the resulting fragments after S9.6 enrichment presumably contain only the RNA:DNA hybrid but not the non-template strand. After denaturing, the template DNA is subjected to 3' ssDNA adaptor ligation, extension, 5' adaptor ligation, PCR and sequencing.
- bisDRIP-seq takes advantage of bisulfite for converting cytosines (Cs) into uracils (Us) within single-stranded DNA regions, and S9.6 for enriching R-loop structures. With subsequent sequencing and analyses, the direction and location of R-loop could be inferred from the distribution of C-to-U mutations. This method was developed by Samie Jaffrey group in 2017 (PMID: 29072160).
- R-ChIP-SS, developed by Xiang-Dong Fu group, basically follows the typical ChIP-seq protocol (PMID: 29104020). Two key differences are introduced, i.e., catalytically-inactive human RNase H1 is exogenously expressed in cells for capturing R-loops in vivo, and the strand-specific sequencing library is prepared with random primer extension on template DNA.
- MapR, following the basic principle of CUT&RUN, utilizes RNase H to guide micrococcal nuclease to R-loop regions for cleavage, release and sequencing. This method was first reported by Kavitha Sarma group (PMID: 31665646), which avoids the step for generating stable cell lines expressing mutant RNase H as is the case for R-ChIP.
- RR-ChIP, a derivative version of R-ChIP reported by Nick Proudfoot group (PMID: 31679819), sequences the RNA moiety instead of template DNA using dUTP strand-specific sequencing strategy.
- qDRIP-seq, similar to ssDRIP, sequences the template DNA in strand-specific manner. However, the isolated DNA is fragmented with sonication instead of enzymatic digestion, and spike-ins might be introduced in for quantitatively measurement of R-loop formation. This method was reported by Karlene Cimprich group in 2020 (PMID: 32544226).
- BisMapR, developed by Kavitha Sarma group in 2021 (PMID: 33620319). R-loops are released by MapR, then treated with non-denaturing bisulfite to convert cytosines on the ssDNA strand. The remaining DNA:RNA hybrid is subjected to second-strand synthesis with dUTP and strand-specific library construction and sequencing.
- R-loop CUT&Tag, based on the principle of CUT&Tag, utilizes hybrid binding domain of RNase H1 to guide Tn5 transposase for adaptor loading, followed by library construction and sequencing. This new method was recently developed by Kaiwei Liang group (PMID: 33597247)
- spKAS-seq, uses N3-kethoxal to label single-stranded DNA in live cells and detects R-loops by measuring asymmetric read density between the two DNA strands. This approach was developed by Chuan He group in 2022 (PMID: 36449625).
- R-ChIP-NS, developed by Tanya T. Paull group in 2024 (PMID: 38717338). The procedure is identical to R-ChIP, except that a non-strand-specific DNA library kit is used instead of a strand-specific protocol.
- RIAN-seq, developed by Hongjie Yao group in 2025 (PMID: 40112807). eGenomic DNA is fragmented with restriction enzymes and treated with nuclease P1, T5 exonuclease, and lambda exonuclease to degrade ssRNA, ssDNA, and dsDNA. The remaining RNA:DNA hybrids are tagmented by Tn5 transposase, followed by PCR library construction.
- enDR3-ChIP, developed by Roman J. Szczesny group in 2025 (PMID: 40823807). enDR3 is an engineered tandem N-terminal hybrid-binding domain of bacterial RNase H3 with point mutations. It is expressed in cells as an mEGFP fusion. After sonication without crosslinking, enDR3-bound chromatin is immunoprecipitated with GFP-trap beads, and purified DNA is used for library preparation and sequencing similar to standard ChIP-seq.
- enDR3-DRIPc-seq, is also developed by Roman J. Szczesny group in 2025 (PMID: 40823807). The same enDR3 protein is used to immunoprecipitate R-loops from sonicated genomic DNA. After DNase I treatment, the remaining RNA is converted to cDNA using a strand-specific dUTP protocol, followed by library preparation and sequencing similar to DRIPc-seq.