Q1: What is ssDRIP-seq?

A1: ssDRIP-seq (single-strand DNA ligation-based DRIP-seq) is a sequencing method for genome-wide R-loop mapping that retains strand information, allowing researchers to distinguish sense and antisense R-loops with high sensitivity.

Q2: How does ssDRIP-seq differ from DRIP-seq?

A2: Conventional DRIP-seq detects R-loops but does not preserve strand-specific details and often suffers from background noise. ssDRIP-seq uses adaptor ligation to maintain strand specificity and produces more accurate, reproducible results with less sequencing depth.

Q3: What types of samples are suitable for ssDRIP-seq?

A3: Cells, tissues, genomic DNA, and IP-enriched DNA are accepted. Minimum inputs are 2 × 10^7 cells, 400 mg tissue, 10 µg genomic DNA, or 50 ng IP DNA.

Q4: What deliverables will I receive?

A4: Clients receive raw sequencing data (FASTQ files), aligned and processed datasets, peak annotations, differential analysis results, and visualisation outputs such as genome browser tracks, motif logos, and pathway enrichment plots.

Q5: What are the typical applications of ssDRIP-seq?

A5: ssDRIP-seq is widely used for studying transcriptional regulation, chromatin organisation, non-coding RNA function, genome stability, and the molecular mechanisms of diseases associated with R-loop accumulation.

ssDRIP-seq Service | R-Loop Mapping & Profiling

Sample Type	Minimum Input	Transport	Storage
Cells	≥ 2 × 10^7	Ship on dry ice in sealed tubes	Store at −80 °C after liquid nitrogen freezing
Tissue	≥ 400 mg	Ship on dry ice in sealed tubes	Store at −80 °C after liquid nitrogen freezing
Genomic DNA	≥ 10 µg	Ship with ice packs	Store at −20 °C (short-term) or −80 °C (long-term), avoid freeze–thaw cycles
IP-enriched DNA	> 50 ng	Ship with ice packs	Store at −20 °C (short-term) or −80 °C (long-term), avoid freeze–thaw cycles

R-loops are RNA:DNA hybrid structures that can influence transcription, chromatin state, and genome stability. Despite their importance, their prevalence, conservation across species, and relationship to chromatin signatures were poorly understood. This study aimed to generate a high-resolution, strand-specific atlas of R-loops in mammalian genomes and link them to functional chromatin states.

The researchers used DRIPc-seq, a strand-specific derivative of DRIP-seq, in human and mouse cells. The method combined S9.6 antibody-based immunoprecipitation with RNA recovery and cDNA conversion, allowing mapping of R-loops with both high resolution and strand specificity. Comparative analyses were performed across multiple cell types and species, including human embryonic carcinoma cells (Ntera2), K562 cells, fibroblasts, and mouse embryonic stem cells. Chromatin accessibility and histone modification datasets (ENCODE) were integrated to correlate R-loop locations with epigenomic states.

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

Conservation: Comparative analysis revealed conserved R-loop formation at promoters and terminators across human and mouse orthologous loci (see Figure 1A

Figure 1. Cluster Analysis of sGB-R-Loops and asTSS-R-Loops during Development.)

Dynamics: Inhibiting transcription initiation (via DRB) showed rapid disappearance of promoter R-loops (half-life ~10 min) and slower turnover of terminator R-loops, supporting their co-transcriptional and dynamic nature.

Chromatin state: R-loops correlated with open chromatin signatures (DNase hypersensitivity, FAIRE signals) and were enriched for active histone marks (H3K4me3, H3K27ac) at promoters, while terminal R-loops overlapped with enhancer- and insulator-like states (see Figure 2 and Figure 3).

Figure 2. Uncoupling between R-Loop Dynamics and RNA Expression Changes.

Figure 3. Regulatory Network and Potential Roles of R-Loops in Regulating Gene Expression.

This study demonstrated that R-loops are:

Prevalent and conserved across mammalian species
Dynamic structures formed co-transcriptionally and resolved within minutes
Associated with distinct chromatin signatures, acting as regulators of promoter activity and transcription termination

These findings position R-loops as integral regulators of gene expression and chromatin organisation, not just accidental by-products.