Is DRIPc-seq strand-specific?

Yes; it sequences the RNA strand in R-loops, enabling strand-specific R-loop profiling with high fidelity

How does DRIPc-seq differ from DRIP-seq?

DRIP-seq maps R-loops via DNA sequencing, but DRIPc-seq sequences the RNA in the hybrid, offering precise strand information

What sample types are compatible with DRIPc-seq?

DRIPc-seq works with diverse samples—cells, tissues, or purified DNA—as long as starting material meets input requirements

Do free RNAs interfere with DRIPc-seq results?

Bulk RNase treatment can reduce signal, but optimized protocols preserve R-loop RNA while removing contaminants

Why choose DRIPc-seq for R-loop mapping?

It offers low background, high resolution, and strand specificity, making it superior to conventional methods for detailed R-loop profiling

DRIPc-seq Service for Strand-Specific R-Loop Profiling

Category	Examples
Animals	Human, mouse, rat, pig, cattle, rhesus monkey, chicken, sheep, honeybee, shellfish, fish, and other tissues or cultured cells.
Plants	Arabidopsis, rice, soybean, maize, tomato, strawberry, rapeseed, grape, apple, hemp, cotton, wheat, peach, tea, tobacco, Asteraceae species, eggplant, magnolia, and more.
Microorganisms	Aspergillus flavus, E. coli, methanogens, actinomycetes, yeast, brown planthopper, whitefly, microalgae, Toxoplasma gondii, bacteria, and fungi.
Plant Tissues	Leaves, seedlings, buds, stems, panicles, callus tissue, fruits, and roots.
Animal Tissues	Blood, embryos, blastocysts, kidney, liver, bladder, cervix, thyroid, pancreas, spleen, thymus, stomach, ovary, mammary gland, skin, muscle, cartilage, colon, nervous tissue, brain, vascular tissue, mucosa, and sponge-like tissue.

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, formed by RNA–DNA hybrids and displaced single-stranded DNA, were historically considered transcriptional by-products. Increasing evidence links them to transcription regulation, chromatin remodeling, and genome instability. The study aimed to profile R-loops at high resolution using DRIPc-seq across human and mouse cells, to determine their prevalence, conservation, and association with chromatin states.

Technique: DRIPc-seq (DNA–RNA immunoprecipitation with cDNA conversion and sequencing) was performed alongside DRIP-seq for comparison.

Samples: Human Ntera2 (embryonal carcinoma) cells and mouse embryonic fibroblasts and stem cells.

Controls: RNase H digestion confirmed that detected signals were genuine RNA–DNA hybrids.

Analyses: Genome-wide profiling of DRIPc-seq peaks, correlation with gene expression (RNA-seq), and integration with epigenomic data (ENCODE histone modifications, DNase hypersensitivity, FAIRE-seq).

Prevalence: R-loops occupied ~5% of the human genome (~70,000 peaks) with a median size of 1.5 kb.

Dynamics: R-loops formed co-transcriptionally and were rapidly resolved after transcription inhibition, supporting dynamic turnover.

Conservation: Strong conservation was observed across human and mouse genomes, particularly at CpG island promoters and transcription terminators.

Chromatin states: Promoter R-loops correlated with open chromatin, CpG islands, H3K4 methylation, and DNA hypomethylation.

Terminal R-loops associated with enhancer- and insulator-like signatures involving CTCF and cohesin.

DRIPc-seq R-loop conservation across species with chromatin correlation. Conserved R-loop formation across human and mouse genomes detected by DRIPc-seq.

DRIPc-seq revealed that R-loops are abundant, dynamic, and conserved genomic features. They preferentially form at promoters and terminators of poly(A)-dependent genes, influencing transcription termination and chromatin structure. Rather than being accidental by-products, R-loops represent a programmed component of mammalian chromatin with regulatory potential.