Table 1.

Techniques for profiling genome-wide R-loop distribution.

	Technique	Innovation	Experimental workflow	Strengths	Weaknesses	Ref.
DRIP-seq and its derivatives	DRIP-seq	Using the intrinsic specificity of the S9.6 antibody for RNA-DNA hybrid	1. DNA extraction 2. Restriction enzyme digestion 3. Immunoprecipitation with S9.6 antibody 4. ds DNA sequencing	Convenient, robust signal	Low resolution, higher background, not strand specific, S9.6 antibody’s bias in sequence recognition [100]	[50, 89]
	RDIP-seq	Pre-treatment with RNase I, use of sonication, directional sequencing	1. Whole cell nucleic acid sonication 2. RNase I pre-treatment 3. Immunoprecipitation with S9.6 antibody 4. RNA sequencing	Reduced background, Strand-specific	A sonication step for nucleic acid fragmentation, which has been shown to reduce the number of genomic R-loops, off target affinity of the S9.6 antibody for dsRNA	[90]
	S1-DRIP-seq	Using optimized levels of S1 nuclease to preserve RNA-DNA hybrid during sonication	1. DNA extraction 2. S1 nuclease digestion 3. Sonication 4. Immunoprecipitation with S9.6 antibody 5. dsDNA sequencing	Quantitative recovery of R loops, High-resolution	Not strand specific, S9.6 antibody’s bias in sequence recognition	[91]
	DRIPc-seq	Further digestion by DNase I, cDNA conversion	1. DNA extraction 2. Restriction enzyme digestion 3. Immunoprecipitation with S9.6 antibody 4. DNase I treatment 5. RNA recovery and reverse transcription to cDNA 6. RNA sequencing	High resolution, strand specific	Off target affinity of the S9.6 antibody for dsRNA	[18, 89]
	ssDRIP-seq	Distinguish specific DNA strands with fewer steps for library construction than DRIPc-seq	1. DNA extraction 2. Sonication 3. Immunoprecipitation with S9.6 antibody 4. ssDNA sequencing	Strand specific	S9.6 antibody’s bias in sequence recognition	[92]
	bisDRIP-seq	In vivo R-loop profiling, combining the use of the S9.6 antibody with sodium bisulfite treatment	1. Cell lysis in the presence of bisulfite 2. DNA extraction 3. Restriction enzyme digestion 4. Immunoprecipitation with S9.6 antibody 5. Bisulfite-modified dsDNA sequencing	Discriminate between the R-loop sequence and the surrounding non-R-loop sequence, high resolution, Strand specific	High sequencing depth is needed, background conversions in ds DNA by bisulfite, S9.6 antibody’s bias in sequence recognition	[93]
	qDRIP-seq	Combining synthetic RNA-DNA hybrid internal standards (spike-in)	1. Adding spike-in to cell lysate 2. Sonication 3. Immunoprecipitation with S9.6 antibody 4. ssDNA sequencing	Accurate cross-condition normalization, absolute quantitation, sensitive, high resolution, strand-specific	S9.6 antibody’s bias in sequence recognition	[56]
Other S9.6 based approaches	DRIP-chip	DRIP followed by hybridization on tiling microarray	1. Crosslinking with formaldehyde 2. Sonication 3. Immunoprecipitation with S9.6 antibody 4. T7 RNA polymerase amplification 5. Biotin labeling 6. Microarray	S9.6 antibody’s bias in sequence recognition		[94]
	S9.6 ChIP-seq (chromatin immunoprecipitation with antibody S9.6, followed by deep sequencing of immunopurified DNA fragments)	Application of ChIP-seq to mapping R loops	1. Crosslinking with formaldehyde 2. Sonication 3. Immunoprecipitation with S9.6 antibody 4. Reverse crosslinking 5. dsDNA sequencing	High-resolution	Formaldehyde crosslinking could affect results, not strand specific	[95]
RNase H based approaches	DRIVE-seq (DNA:RNA in vitro Enrichment)	Using specificity of catalytically dead RNase H1 for RNA-DNA hybrid	1. Genomic DNA extraction 2. Restriction enzyme digestion 3. Catalytically dead RNase H1 incubation 4. Pull-down 5. dsDNA sequencing	Enable the specific and near quantitative recovery of R loop molecules in complexnucleic acid mixture exquisite specificity of RNase H	Low capture efficiency, not strand specific, substantial RNase H-resistant regions on the genome	[50]
	R-ChIP	In vivo R-loop profiling using catalytically dead RNase H1	1. Introduce V5-tagged catalytically dead mutant RNase H1 into cells 2. Sonication 3. Immunoprecipitation with anti-V5 antibody 4. ssDNA sequencing	exquisite specificity of RNase H, strand-specific	substantial RNase H- resistant regions on the genome, require the generation of stable cell lines (time-consuming)	[34, 96]
	MapR	Combining the specificity of RNase H for RNA-DNA hybrid with CUT&RUN approach	1. Immobilize cells on beads 2. Incubate with GST-RNaseHΔcat-MNase 3. R-loop recognition by RNaseHΔcat 4. MNase mediated R-loop cleavage 5. DNA sequencing	Antibody-independent, does not require the generation of stable cell lines (fast and convenient), high sensitivity with low input material	substantial RNase H-resistant regions on the genome	[97, 98]
	R-loop imaging	Imaging and quantifying R-loops using GFP-catalytically dead RNase H1 (dRNH1)	1. Expression and purification of GFP-dRNH 2. Transfection in fixed cells 3. Imaging	Using purified GFP-dRNH1 protein, bypassing the need for cell line engineering	GFP-dRNH1 has its own limitations (non-specific binding, binding preference to G-rich)	[99]