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.
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Technology Introduction
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.
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.
Map R-loops at TSS/TES, CpG/GC-rich regions, and gene bodies to study RNAPII pausing, termination, and enhancer–promoter activity.
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.
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.
Tunable for plant, microbial, and mammalian genomes (e.g., TAIR10, hg38, mm10), enabling comparative R-loop landscapes.
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?
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 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.
- Annotation: Nearest genes, promoter/gene body/intergenic class, GC-rich island overlap, TSS/TES proximity.
- Signal Tracks: bigWig (sense/antisense) for IGV/UCSC/JBrowse; CPM/RPM normalized; spike-in normalized when selected.
- Differential Analysis: Condition contrasts with log2FC, FDR/q-values, effect sizes, and concise result tables.
- Figures & Report: Browser snapshots, volcano/MA, metagene profiles, heatmaps, and a clean study report ready for Methods sections.
Optional Add-Ons
- Functional Enrichment: GO/KEGG on differential R-loop genes (strand-aware subsets on request).
- Motif Discovery: Sense vs antisense motif analysis; TF motif overlap at R-loop hotspots.
- R-Loop Cluster Hotspots: Detect clustered R-loops and summarize hotspot burden per locus/condition.
- Genome Stability Context: Overlap with γH2AX/repair marks or replication features (when data available).
- Multi-Omics Integration: Joint views and statistics with RNA-seq, ChIP-seq (RNAPII/TFs), ATAC-seq.
- Delivery Extras: UCSC track hubs, IGV session files, custom genome builds.
Ready To Customize Your Analysis?
Request our analysis menu or share your hypothesis—we'll tailor the pipeline to your organism, contrasts, and downstream assays.
What You'll Receive
- Data Files: FASTQ · BAM/BAI · bigWig (sense/antisense) · Peak BED/narrowPeak (by strand/condition).
- 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
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 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.
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)
- Prefer in-cell capture (sensor-based) or limited input? → R-ChIP / R-loop CUT&Tag
- Baseline/global profiling without strand info? → DRIP-seq
Compact Comparison (Essentials Only)
| Method | Strand Information (DNA/RNA) | Best For | Caveats |
|---|---|---|---|
| ssDRIP-seq | DNA (sense/antisense) | Strand-aware DNA:RNA hybrid mapping; differential studies with RNase H control | Requires S9.6 IP; input per SOP |
| DRIP-seq | None | Legacy, broad hybrid profiling | Lower resolution; no strand context |
| DRIPc-seq | RNA | Identifying RNA species in hybrids | 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
- Consult & Design — Define organism, contrasts, replicates; set controls (RNase H default, spike-ins optional).
- Sample Intake & QC — Verify inputs; preserve native DNA:RNA hybrids.
- Library Prep — S9.6 IP → strand-specific ligation (3′/5′) → indexed PCR.
- Sequencing — Illumina paired-end configuration agreed at design.
- Analysis & Delivery — Strand-aware peaks (BED), bigWig tracks (sense/antisense), BAM, QC, and a concise report; optional review call.