Get a Quote
quote
Get a Quote
×
Quote Request

High-Resolution R-ChIP Service: Antibody-Free R-Loop Mapping

Achieve the highest specificity in R-loop mapping with our R-ChIP (RNase H1 Chromatin Immunoprecipitation) service. By utilizing catalytically inactive RNase H1 (dRNase H1) instead of antibodies, we capture RNA-DNA hybrids in vivo with near-zero background from dsRNA. Ideal for validating DRIP-seq results and mapping R-loops at high resolution. RUO.

  • Antibody-free capture using dRNase H1 (D210N)
  • Eliminates S9.6 cross-reactivity with dsRNA
  • High-resolution mapping of R-loop boundaries
  • Strand-specific output for sense/antisense resolution
Discuss Your Project

3D illustration of dRNase H1 binding to an R-loop structure in R-ChIP sequencing.

Overview: Precision R-Loop Profiling with dRNase H1

R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a displaced single strand of DNA. While these structures play essential roles in gene regulation, chromatin patterning, and DNA repair, their unscheduled accumulation is a hallmark of genomic instability and replication stress. Our High-Resolution R-ChIP Service (RNase H1 Chromatin Immunoprecipitation) provides a robust, "research-grade" solution to map these structures across the entire genome with superior specificity compared to traditional antibody-based methods.

Standard R-loop mapping methods (like basic DRIP-seq) utilize the S9.6 antibody, which is known to bind double-stranded RNA (dsRNA) in addition to RNA-DNA hybrids, leading to potential artifacts. Our service solves this by using a catalytically inactive RNase H1 (dRNase H1) mutant as an affinity probe. This protein binds tightly and specifically to RNA-DNA hybrids in vivo without degrading them, allowing for the capture of native R-loops with near-zero background. This approach distinguishes between "regulatory" R-loops (often promoter-associated) and "deleterious" R-loops (replication blocks), providing a clear, actionable picture of genomic health.

Service Snapshot

  • Target: DNA-RNA Hybrids (R-loops)
  • Probe: V5-tagged dRNase H1 (D210N mutant)
  • Specificity: High (No dsRNA cross-reactivity)
  • Readout: Strand-specific NGS peaks (Sense/Antisense separation)

Why R-ChIP Beats Standard Antibody Methods?

Specificity by Design

The dRNase H1 protein has evolved biologically to recognize only the structural features of RNA-DNA hybrids. Unlike antibodies that can be "sticky," this enzyme-based capture acts as a molecular filter, naturally excluding dsRNA and ssDNA artifacts that plague other methods.

Strand-Specific Mapping

We employ a dUTP-based strand-specific library preparation method. This marks the second strand of DNA during synthesis, allowing the sequencer to distinguish the original template strand. This lets you determine if the R-loop RNA matches the gene's coding strand (sense) or arises from antisense transcription.

High-Resolution Peak Calling

Because dRNase H1 is a small enzyme that binds directly to the hybrid structure, the resulting data (peaks) are much sharper and narrower than those obtained from antibody immunoprecipitation. This improved resolution allows researchers to distinguish distinct R-loop boundaries, such as those forming exactly where RNA Polymerase II pauses.

Comprehensive Bioinformatics

We don't just find peaks; we contextualize them. Our bioinformatics pipeline includes rigorous quality control, peak calling against appropriate backgrounds, and annotation to genomic features (promoters, gene bodies, terminators) and sequence motifs (G-richness).

Technical Comparison: R-ChIP vs. DRIP-seq vs. MapR

Feature R-ChIP (Our Service) DRIP-seq MapR
Capture Method dRNase H1 Protein (In vivo expression) S9.6 Antibody (In vitro on gDNA) Recombinant RNase H (In situ diffusion)
Specificity Very High (Zero dsRNA binding) Moderate (Risk of dsRNA binding) High
Resolution High (Sharp peaks) Low (Broad domains) High (Enzyme tethered)
Sample Prep Requires Cell Transfection Genomic DNA (No transfection) Permeabilized Cells/Nuclei
Best For High-specificity validation & Mechanism Discovery screens & Clinical samples Low-input samples & Speed
Key Limitation Not suitable for difficult-to-transfect cells Requires RNase H negative controls Requires careful enzyme titration

Workflow – Step-by-Step R-ChIP Procedure

The R-ChIP workflow relies on expressing the probe within the cell itself. We handle the entire process from vector construction to bioinformatic delivery.

1. Plasmid Construction & Transfection

We utilize a validated expression vector containing the V5-tagged dRNase H1 (D210N) sequence. Your cells are transfected (Lipofectamine or Electroporation) to express the probe. QC: Western blot to confirm expression.

2. Protein Expression & Binding

Cells are cultured for an optimized period (typically 24-48 hours) to allow the dRNase H1 protein to be produced, enter the nucleus, and bind tightly to endogenous R-loops.

3. Crosslinking & Chromatin Prep

We treat the cells with formaldehyde to "lock" the protein-DNA complexes in place, preserving the in vivo interactions. Nuclei are isolated and chromatin is fragmented (sonication) to the optimal size range (200-500 bp).

4. Tag-Based Immunoprecipitation

We use high-affinity anti-V5 magnetic beads to pull down the dRNase H1 protein—and crucially, the DNA it is holding onto. This tag-based purification is cleaner than standard antibody IP.

5. Strand-Specific Library Prep

We prepare sequencing libraries using a dUTP-based method that preserves the direction of the DNA strands. This ensures we can resolve sense from antisense R-loops.

6. Sequencing

Libraries are sequenced on the Illumina NovaSeq platform (PE150) to a depth sufficient to detect both abundant and rare R-loop events (>40 million reads).

7. Bioinformatics Analysis

Mapping reads to reference genome, Peak Calling using specialized algorithms, and Annotation to genomic features.

Step-by-step R-ChIP service workflow from transfection to sequencing.

Technical Specifications

We offer flexible formats to match your resolution and throughput needs.

Specification Details
Probe V5-tagged dRNase H1 (D210N Mutant)
Library Prep Strand-Specific (dUTP method) to preserve directionality.
Sequencing Depth Standard: 40-50 Million PE150 reads per sample.
Resolution ~200-500 bp (Sonication based).
Input Requirements Recommended: 10 million transfectable cells.
Controls Input chromatin (or mock transfected control if requested).

Recommended Applications of R-ChIP

Understanding where and why R-loops form is critical for research into cancer biology, neurodegeneration, and basic gene control.

Mechanisms of Genomic Instability

R-loops are a major source of DNA damage. When the replication machinery collides with an R-loop, it can cause DNA breaks (DSBs). By mapping these collision sites with R-ChIP's high resolution, researchers can study Transcription-Replication Conflicts (TRCs) and how cells maintain genome stability under stress.

Transcriptional Pausing and Termination

R-loops naturally form at the ends of genes to help stop transcription (termination) or at promoter regions to pause the machinery. Our service maps these "healthy" R-loops to study how genes are turned on and off and how promoter-proximal pausing is regulated.

Validating Drug Targets

Drugs that target DNA Damage Response (DDR) pathways—such as PARP inhibitors—often cause R-loops to accumulate. R-ChIP provides the specific evidence needed to prove that a drug is working by stabilizing R-loops at expected loci, rather than just causing general stress.

Disease Modeling

In diseases like Cockayne Syndrome or Fragile X, R-loop metabolism is perturbed. R-ChIP allows for the comparison of R-loop landscapes between healthy and diseased cells, pinpointing the exact genes where regulation fails.

Why Choose CD Genomics for R-ChIP?

R-Loop Specialists

We have extensive experience with all major R-loop mapping technologies (DRIP, R-ChIP, MapR) and can guide you to the right choice.

Optimized Vectors

We use validated expression constructs that ensure proper nuclear localization and binding of the dRNase H1 probe.

Rigorous QC

We perform stringent quality checks at every step, from transfection efficiency to library complexity, ensuring your data is reliable.

Bioinformatics Expertise

Our team understands the nuances of R-loop data, including how to filter artifacts and interpret strand-specific signals.

Sample Requirements

Sample Type Requirement Notes
Transfectable Cell Lines >10 million cells (High Viability) Cells must be amenable to transfection (Lipofectamine or Electroporation). HEK293, HeLa, U2OS work best.
Stable Cell Lines >10 million cells If you already have a stable line expressing V5-dRNaseH1, we can start directly from crosslinking.
Primary Cells / Tissue Not Recommended Primary tissues cannot be easily transfected. For these, we recommend MapR or DRIP-seq.
Shipping Frozen Cell Pellet or Live Culture Consult our team for specific shipping buffers to preserve viability.

Key Deliverables

We provide comprehensive data packages designed to answer your biological questions immediately.

Raw Data Files
FASTQ files (Clean reads).

Signal Tracks (BigWig)
Forward Strand (Blue) for sense R-loops and Reverse Strand (Red) for antisense R-loops.

Peak Files (BED)
Precise genomic coordinates of significantly enriched R-loops.

Metagene Profiles
Aggregate plots showing R-loop enrichment patterns (e.g., TSS/TES peaks).

Case Study: R-ChIP Uncovers Transcription-Associated R-Loops in Cockayne Syndrome

Cockayne Syndrome (CS) is a severe genetic disorder linked to defects in DNA repair. In a 2024 study, researchers investigated whether the loss of the CSB protein caused R-loops to accumulate, leading to DNA damage. They needed a high-specificity method to map these R-loops without the noise associated with antibody methods.

The team used R-ChIP by expressing V5-tagged dRNase H1 in human cells. They compared control cells to cells where CSB was knocked down. They integrated this data with PRO-seq (which maps active RNA Polymerase) to see exactly where transcription was stalling.

R-ChIP profiling revealed a global increase in R-loops in the CSB-deficient cells. Crucially, the high resolution of R-ChIP allowed them to see that these R-loops were forming specifically at sites where RNA Polymerase II was "stalled" or stuck. This provided a direct mechanistic link: without CSB, the polymerase gets stuck, R-loops form, and the genome becomes unstable.

R-ChIP data showing genome-wide R-loop distribution in Cockayne Syndrome models.

The use of R-ChIP provided the specific, high-resolution evidence needed to link a transcription defect to R-loop accumulation, offering a new model for the disease.

(Source: Cockayne Syndrome Linked to Elevated R-Loops, Nature Communications, 2024. CC BY 4.0)

Demo Results (Representative Examples)

  • IGV genome browser tracks comparing R-ChIP resolution against DRIP-seq data.
  • Annotated peak files mapping R-loops to promoters and gene bodies.
  • Metagene plots showing global R-loop distribution relative to TSS.
  • QC reports validating dRNase H1 expression and enrichment efficiency.

IGV genome browser tracks comparing R-ChIP resolution against DRIP-seq data.IGV Tracks

Metagene profile showing R-loop enrichment at TSS/TES.Metagene Profile

Differential R-loop analysis volcano plot.Differential Analysis

Frequently Asked Questions

References

  1. Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation. Nature Communications. 2024.
  2. A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide. eLife. 2021.
  3. Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq. eLife. 2017.
Leading Your Research Forward

Enhancing your vision research capabilities.

High-confidence 3D genomics services for chromatin interaction analysis and regulatory insight.

Quick Links
Contact Us
Copyright © CD Genomics. All Rights Reserved.
Top