ssDRIP-Seq Service — Strand-Specific R-Loop Mapping For Research
A New Standard In Strand-Specific R-Loop Profiling
Profile genome-wide DNA:RNA hybrids with ssDRIP-seq (single-strand DNA ligation–based DRIP-seq) for clear, reproducible, strand-aware maps—validated with RNase H.
Built on S9.6 enrichment, strand-specific library preparation, and Illumina sequencing, our service delivers:
Strand-separated peaks and bigWigs (sense/antisense)
RNase H negative control included by default
Optional DNA:RNA hybrid spike-ins for quantitative comparisons
Sequence-independent ligation to reduce bias
End-to-end workflow and an interpretable bioinformatics report
At CD Genomics, we enable researchers to probe transcription regulation, replication stress, and genome stability with analysis-ready R-loop data—for research use only.
ssDRIP-Seq (Single-Strand DNA Ligation–Based DRIP-Seq) is a strand-specific R-loop mapping method that enriches DNA:RNA hybrids with the S9.6 antibody and preserves which DNA strand forms the hybrid through single-strand adapter ligation. The result is a genome-wide, strand-aware atlas of R-loops suitable for mechanistic studies of transcription regulation, replication stress, and genome stability—for research use only.
How The Assay Works—At A Glance
Enrichment: Mild extraction and restriction fragmentation followed by S9.6 immunoprecipitation of DNA: RNA hybrids.
Strand Preservation: 3′/5′ single-strand ligation (sequence-independent) retains sense/antisense information and minimizes primer bias.
Validation: RNase H control (default) verifies hybrid specificity and improves peak-calling thresholds.
Sequencing & Analysis: Illumina paired-end sequencing with a strand-aware pipeline producing peaks, tracks, and an interpretable report.
Why Researchers Choose ssDRIP-Seq
Strand-Specific R-Loop Mapping: Identify promoter/terminator hotspots and GC-rich regions with sense vs antisense context.
Higher Specificity & Reproducibility: S9.6 IP plus RNase H reduces off-target signals and improves replicate concordance.
Designed For Comparisons: Optional DNA: RNA hybrid spike-ins support quantitative between-group analyses (mutants, treatments, timepoints).
Integration-Ready Outputs: BigWig tracks and peak sets that align seamlessly with RNA-seq, ChIP-seq, and ATAC-seq.
Applications
What Is ssDRIP-seq Used For
Use ssDRIP-seq when you need strand-specific, genome-wide evidence of where and how DNA:RNA hybrids form—and how they change.
Promoter & Terminator Hotspots
Map R-loops at TSS/TES, CpG/GC-rich regions, and gene bodies to study RNAPII pausing, termination, and enhancer–promoter activity.
Differential R-Loop Profiling
Compare mutants, treatments, or timepoints; add spike-in normalization for quantitative burden shifts across conditions.
Overlay strand-aware peaks with γH2AX/repair marks, replication features, or TOP1/SETX/FANCD2 pathways to flag instability hotspots.
Antisense & Noncoding RNA Programs
Resolve sense vs antisense R-loops; optionally pair with DRIPc-seq to identify the RNA species, or integrate with RNA-seq to connect hybrids to expression changes.
Cross-Organism Studies
Tunable for plant, microbial, and mammalian genomes (e.g., TAIR10, hg38, mm10), enabling comparative R-loop landscapes.
Regulatory Element Prioritization
Integrate with ChIP-seq/ATAC-seq to rank candidate promoters/enhancers by strand-aware hybrid signal and motif context.
Typical Questions We Help Answer
Does my mutation increase R-loops at promoters or within the gene body?
Does a candidate drug reduce global R-loop burden or shift it to specific loci?
Which loci show strand-biased hybrids consistent with my TF or chromatin mark profiles?
Workflow
How It Works
End-To-End ssDRIP-seq Pipeline — designed to preserve native DNA:RNA hybrids, keep strand information, and deliver clean, comparable signal.
Sample Intake & Preservation Of R-Loops
Mild extraction from cells/tissues or purified gDNA to retain native DNA:RNA hybrids. Basic QC (quantity, purity) before proceeding.
Restriction Fragmentation
Enzyme-based fragmentation under gentle conditions to generate DRIP-compatible fragments while maintaining hybrid structures for downstream capture.
S9.6 Immunoprecipitation (Enrichment)
The S9.6 antibody selectively enriches DNA:RNA hybrids. Stringent washes minimize background and improve downstream FRiP and replicate concordance.
RNase H Negative Control (Default)
A parallel aliquot is treated with RNase H to digest DNA:RNA hybrids. This control validates specificity, informs peak thresholds, and improves false-positive control.
Strand-Specific Library Construction
Single-strand adapter ligation on the captured ssDNA: 3′ adapter → 5′ adapter, followed by indexed PCR. This sequence-independent ligation preserves sense/antisense orientation and reduces primer bias.
Illumina Paired-End Sequencing
Paired-end reads support accurate alignment and strand-aware coverage profiles. Depth is tuned to genome size and study goals (set during consult).
Primary Processing & Delivery
Demultiplex → alignment → duplicate handling → strand-aware peak calling → normalization (CPM; spike-in if selected) → export of bigWig tracks, peak BEDs, QC, and a concise report.
(See "Bioinformatics And Add-Ons" and "What You'll Receive" for full deliverables.)
Bioinformatics
Bioinformatics And Add-Ons
Turn your ssDRIP-seq reads into strand-aware, interpretable biology—without extra lift from your team.
Standard Analysis
Read QC & Alignment: FastQC/MultiQC, strand-aware alignment, duplicate handling.
Peak Calling (Strand-Specific): Sense/antisense peaks with RNase H–validated thresholds; FRiP and enrichment metrics reported.
Results Tables: Differential R-loop peaks (log2FC, FDR), gene/feature annotations.
QC Summary: Read/library metrics, IP enrichment, FRiP, replicate concordance, RNase H check.
Figures: Browser snapshots and key plots (volcano/MA, metagene/heatmap) for quick review.
Report: Methods, parameters, and concise findings—ready for Materials & Methods.
Delivery: Structured folders via secure link/SFTP, with checksums; optional IGV session or UCSC track hub.
KEGG Pathway Analysis of Differentially Enriched Regions
GO Enrichment Analysis of Differentially Enriched Regions
Sense/Antisense Peak Motif Analysis
Sense/Antisense Chromosomal Distribution of Peaks
Sequencing Plan
Sequencing Plan
Platform & Configuration
Illumina Paired-End (Default): Paired-end reads improve alignment accuracy, fragment-size estimation, and strand-aware coverage for ssDRIP-seq.
Read Length: PE150 recommended for most genomes; shorter reads possible for cost-sensitive designs.
Single-End (Optional): Available on request; we still recommend paired-end for peak resolution and QC robustness.
Depth Guidelines (Set During Consult)
Large Genomes (Human/Mouse/Comparable): Aim for moderate–high depth to resolve promoter/terminator hotspots and call differential R-loop peaks confidently.
Mid-Size Genomes (Plants/Yeast): Depth scaled to assembly size and repeat content for stable FRiP and reproducible peaks.
Small Genomes (Bacteria/Organelle): Lower depth typically suffices; we adjust for GC bias and targeted hypotheses.
Comparative Studies: Increase depth for multi-condition contrasts or subtle effect sizes; enable spike-in normalization when quantitative between-group comparisons are critical.
Run And QC Considerations
Balanced Lanes: Libraries randomized across lanes/batches when applicable.
Insert Size Targets: Tuned to assay chemistry for consistent peak width and mapping rates.
Reference Builds: Your choice of hg38, mm10, TAIR10, custom assemblies; we align all deliverables (BAM, bigWig, BED) to the specified build.
Not Sure How Much You Need?
Share your organism, conditions, and number of replicates—we'll return a depth plan that balances sensitivity, FRiP, and budget, optimized for strand-specific R-loop mapping.
Sample
Sample Requirements
Provide clean, intact material to preserve native DNA:RNA hybrids for ssDRIP-seq.
Accepted Types & Minimum Input
Sample Type
Minimum Input
Cells
≥ 2 × 10^7
Tissue
≥ 400 mg
gDNA
≥ 10 μg
Post-IP DNA
≥ 50 ng
Preparation
RNase-free; no added DNase/RNase.
Inhibitor-free buffers; avoid phenol/guanidinium/SDS and residual ethanol.
gDNA intact (A260/280 ~1.8–2.0); do not over-shear or crosslink.
Aliquot to reduce freeze–thaw; 1.5 mL tubes/cryovials sealed.
Shipping & Storage
Ship: Dry ice (DNA may use cold packs if needed).
Store: Cells/tissue snap-freeze, −80 °C; DNA short-term −20 °C. Minimize freeze–thaw.
Intake Info
Organism and reference build (e.g., hg38, mm10, TAIR10).
Planned contrasts/replicates; note any challenging matrices.
Method Selector
Choosing The Right Method
Pick the assay that matches your biological question. If you need strand-specific DNA-side mapping with robust controls, choose ssDRIP-seq. If you need to identify the RNA component of R-loops, pair with DRIPc-seq. For in-cell capture or low input, consider R-ChIP or R-loop CUT&Tag.
Quick Selection Guide
Need DNA strand context (sense/antisense) at hotspots? → ssDRIP-seq
Want to know which RNA forms the hybrid? → DRIPc-seq (can be coordinated with ssDRIP-seq)
RNA-side view; pair with DNA-side mapping for full picture
R-ChIP
DNA (sensor in vivo)
In-cell capture; native context
Requires expression of inactive RNase H1; setup-dependent
R-loop CUT&Tag
DNA (sensor/antibody)
Low-input, native profiling
Sensitivity varies; protocol nuance
Not sure which path fits your hypothesis?
Share organism, contrasts, and sample constraints—we'll recommend the most informative, cost-effective design (and coordinate multi-assay projects when helpful).
Project Workflow
Project Workflow
Consult & Design — Define organism, contrasts, replicates; set controls (RNase H default, spike-ins optional).
KEGG Pathway Analysis of Differentially Enriched Regions
GO Enrichment Analysis of Differentially Enriched Regions
Sense/Antisense Peak Motif Analysis
Sense/Antisense Chromosomal Distribution of Peaks