How does DRIP-Seq detect R-loops genome-wide?

By using the structure-specific S9.6 antibody to immunoprecipitate DNA:RNA hybrids, followed by next-generation sequencing and alignment to the genome, DRIP-Seq reveals R-loop locations.

What makes DRIP-Seq different from DRIPc-Seq?

DRIP-Seq maps where R-loops occur; DRIPc-Seq sequences the RNA strand of the hybrid for strand-specific insights.

Why are R-loops biologically important?

They regulate transcription initiation and termination, modulate chromatin structure, aid in class switch recombination, and unresolved R-loops can cause genome instability.

What are common uses of DRIP-Seq data?

Researchers use DRIP-Seq to examine R-loop distribution at CpG island promoters, transcription start or end sites, and to study the effects of mutations in R-loop resolving enzymes.

How is DRIP-Seq similar to ChIP-Seq?

Both rely on immunoprecipitation of nucleic acid-protein or structure complexes followed by high-throughput sequencing, aligning to known genome locations.

Why is resolution limited in standard DRIP-Seq?

Because antibody capture enriches broad DNA:RNA hybrid regions, it lacks base-pair resolution, which is improved in variants like bisDRIP-Seq and sDRIP-Seq.

Can DRIP-Seq results vary by fragmentation method?

Yes—sonication provides more uniform and reproducible fragment profiles than restriction enzyme digestion, which may bias coverage.

Is DRIP-Seq applicable across organisms?

Absolutely—it has been successfully applied in humans, yeast, Drosophila, and other systems, and CD Genomics supports diverse sample types like tissues and microbial genomes.

How reliable are DRIP-Seq annotations?

Tools like DROPA improve accuracy by reducing false positives and enhancing gene-level annotation by incorporating gene expression data

DRIP-Seq Service – High-Resolution R-Loop Detection Pipeline

Parameter	Specification
Input DNA requirement	≥1–5 µg high-quality genomic DNA
Sample types	Cells, tissues, plants, and microbial genomes
Antibody	S9.6 monoclonal antibody for DNA:RNA hybrid capture
DNA fragmentation	Sonication-based shearing (optimised for peak resolution)
Sequencing platform	Illumina (paired-end, 150 bp reads)
Recommended depth	30–50 million reads per sample (adjustable for project size)
Quality control	Positive/negative controls, library validation, duplicate removal
Data outputs	Raw FASTQ files, peak annotation tables, genome browser tracks, full reports

Feature / Method	DRIP-Seq	DRIPc-Seq	dsRIP-Seq	R-loop Cut&Tag
Genome-wide coverage	✔	✔	✔	✔
Strand specificity	✘	✔ (detects RNA strand origin)	✘	✔
Input requirement	High (≥1–5 µg DNA)	Medium (similar to DRIP-Seq)	High (≥1–5 µg DNA)	Low (suitable for rare samples)
Sensitivity	High	High	Very High (for double-stranded hybrids)	Medium
Best suited for	General R-loop mapping	RNA strand identification and transcriptional origin	Double-stranded hybrid profiling	Low-input R-loop profiling

Sample Type	Recommended Quantity	Purpose	Notes
Cells	≥ 2 × 10⁸ cells	R-loop profiling via DRIPc-seq	Ensures sufficient hybrid capture and reproducibility
Tissue	≥ 100 mg	R-loop profiling via DRIPc-seq	Adequate material for enzymatic fragmentation and IP
Genomic DNA	≥ 30 µg	Direct input into library prep	Must be high-quality—OD260/280 around 1.8–2.0, DNA-free
Other Formats	Upon consultation	Customized experimental designs	CD Genomics can adapt protocols based on sample type, as needed

R-loops form when nascent RNA hybridizes to its DNA template, leaving the non-template strand displaced. In bacteria, excessive R-loops compromise genome stability and cell viability. Topoisomerase I (EcTopoI) is hypothesized to counteract transcription-induced negative supercoiling and thereby prevent R-loop accumulation.

Researchers used ChIP-Seq, Topo-Seq, and strand-specific DRIP-Seq in E. coli to map EcTopoI binding, cleavage activity, and R-loop distribution under various conditions:
Wild-type cells vs. mutants with truncated or inactive EcTopoI.
Overexpression of a 14 kDa EcTopoI C-terminal domain (CTD) to disrupt EcTopoI–RNA polymerase (RNAP) interaction.
Rifampicin treatment to inhibit RNAP elongation.

Genome-wide colocalization: EcTopoI was enriched upstream of highly transcribed units and colocalized with RNAP across transcription units (Fig. 1, p.3).
Disrupted interaction: Overexpression of the CTD impaired RNAP–EcTopoI coupling, leading to excessive negative supercoiling and hypercompacted plasmid DNA (Fig. 5g, p.9).
R-loop accumulation: DRIP-Seq confirmed genome-wide R-loop build-up when EcTopoI function was disrupted (Fig. 5h, p.10).
Toxicity: Cells with disrupted RNAP–EcTopoI interactions displayed filamentation, growth defects, and SOS-response activation.

DRIP-Seq plot showing increased R-loops in E. coli after TopoI disruption. Strand-specific DRIP-Seq reveals genome-wide R-loop accumulation in E. coli upon disruption of RNAP–TopoI interaction.

This study demonstrates that the direct interaction between RNAP and EcTopoI is essential for suppressing harmful R-loops. By relieving negative supercoiling, EcTopoI safeguards transcriptional fidelity and cell viability. The findings highlight the importance of topoisomerase–polymerase coordination as a genome-stabilizing mechanism in prokaryotes