ChRD-PET Sequencing Service for RNA–DNA Interaction and 3D Genome Profiling

Chromatin-associated RNA–DNA interactions followed by paired-end-tag sequencing (ChRD-PET) offers a new dimension in 3D genomic analysis.

At CD Genomics, we provide an end-to-end ChRD-PET sequencing service that reveals how coding and noncoding RNAs shape chromatin structure and transcriptional regulation.

Our comprehensive workflow—from sample preparation and chromatin immunoprecipitation (ChIP) to sequencing and advanced bioinformatics—helps researchers explore promoter-centered, enhancer-linked, and R-loop-derived RNA–DNA interactions with high precision.

We help clients:

• Map RNA–DNA interactions with high spatial resolution.
• Characterize promoter and enhancer networks driving gene regulation.
• Integrate R-loop and chromatin domain data for multi-layered insights.
• Combine results with ChIA-PET, HiChIP, HiFi-C and Pore-C sequencing for complete 3D genomic understanding.
• Apply the method to plants and other eukaryotes using validated histone antibodies.

• RNA–DNA Interaction Mapping at Single-Molecule Resolution
• Promoter and Enhancer-Centered Chromatin Network Profiling
• R-loop and Noncoding RNA Integration
• Compatible with 3D Genomic Sequencing Platforms

At a glance:

• What Is ChRD-PET?
• How ChRD-PET Works: Workflow Overview
• Key Technical Advantages of ChRD-PET
• Applications of ChRD-PET
• Bioinformatics
• Deliverables
• Sample Requirements
• FAQs

What Is ChRD-PET?

Chromatin-associated RNA–DNA interactions followed by Paired-End Tag sequencing (ChRD-PET) is a cutting-edge technique designed to map how RNAs physically interact with chromatin across the genome. In this workflow, chromatin complexes enriched by specific antibodies (such as H3 or H3K4me3) are ligated using a biotin-labelled DNA bridge linker to spatially adjacent RNA and DNA molecules. The resulting ligated RNA–DNA fragments are sequenced via paired-end sequencing, enabling detection of RNA–DNA contacts with high specificity and resolution.

Originally developed in rice (ChRD-PET was first described in Nature Plants) the method revealed three classes of RNA–DNA interaction: local (cis), proximal (nearby) and distal (often trans-chromosomal).

Why this matters for 3D genome sequencing and functional genomics:

• Unlike standard DNA–DNA chromatin conformation assays, ChRD-PET reveals the RNA dimension of nuclear architecture—how chromatin-associated RNAs (caRNAs) guide or respond to chromatin loops and domains.
• It links transcriptome data with structural genome data, enabling studies of noncoding RNAs, R-loop formation and promoter/enhancer interactions in a single workflow.

By integrating with complementary platforms (such as our 3D genome tools), clients can gain a holistic view of genome regulation—from sequence to structure to RNA.

In our CRO service offering, the ChRD-PET platform is ideal for researchers, academic institutions, biotech and pharma teams who aim to:

• Map RNA–DNA interactomes around active promoters and enhancers.
• Investigate the role of noncoding RNA in chromatin architecture and gene regulation.
• Combine caRNA mapping with 3D genome data (loops, domains) to drive discovery in fields such as crop breeding, epigenetics, drug screening and functional genomics.

How ChRD-PET Works: Workflow Overview

ChRD-PET combines chromatin immunoprecipitation (ChIP), spatial ligation, and paired-end sequencing to capture chromatin-associated RNA–DNA interactions with high specificity. The process follows a clear, reproducible pipeline optimised for 3D genomic analysis.

1. Chromatin Crosslinking

Cells or tissues are fixed with formaldehyde to preserve native RNA–DNA–protein interactions within the nucleus. This ensures spatial relationships between chromatin and RNA are retained during extraction.

2. Chromatin Immunoprecipitation (ChIP) Enrichment

Antibodies specific to histone marks or chromatin-bound proteins (e.g., H3, H3K4me3) selectively enrich target regions. This step defines the genomic context—promoters, enhancers, or specific regulatory domains—where RNA-DNA interactions are captured.

3. Bridge-Linker Ligation

A biotin-labelled DNA bridge linker is introduced to join spatially adjacent RNA and DNA molecules. This unique ligation design eliminates random contact artefacts and enables high-confidence mapping of RNA–DNA proximity events.

4. Reverse Crosslinking and Purification

Crosslinks are reversed, and RNA–DNA chimeric fragments are purified. Only ligated RNA–DNA molecules are retained for sequencing, improving specificity and downstream analysis accuracy.

5. Paired-End Sequencing

The ligated complexes are converted into sequencing libraries and processed on Illumina or comparable high-throughput platforms. Paired-end sequencing enables precise detection of both RNA and DNA tags in each interaction pair.

6. Bioinformatics Reconstruction

Our analytical pipeline aligns paired tags to the reference genome, identifies interaction pairs, and visualises promoter–enhancer networks. Integration with R-loop and chromatin-loop data reveals multi-layered 3D genome organisation.

Together, these steps allow single-molecule-resolution profiling of RNA–DNA interactions, providing essential insights into transcriptional regulation and chromatin topology.

Key Technical Advantages of ChRD-PET

High Sensitivity and Specificity

ChRD-PET detects low-abundance chromatin-associated RNAs (caRNAs) and distinguishes true RNA–DNA interactions from random ligations. The use of a biotin-labelled bridge linker and stringent immunoprecipitation reduces background noise, producing high-confidence interaction maps.

Single-Molecule Resolution

Each paired-end tag represents one RNA–DNA contact event. This direct-read strategy provides quantitative, molecule-level insight into transcriptional regulation and RNA-mediated chromatin organisation.

Promoter-Centred and Enhancer-Linked Mapping

The platform precisely identifies RNA–DNA interactions associated with active promoter and enhancer regions (e.g., H3K4me3-marked sites). It enables researchers to examine how RNAs regulate nearby and distant genes across chromatin loops.

R-Loop and Noncoding RNA Detection

ChRD-PET supports the identification of R-loop–derived RNA–DNA hybrids and the classification of noncoding RNAs (lncRNAs, snoRNAs, miRNAs) involved in chromatin regulation. This expands understanding beyond coding RNAs to the full regulatory RNA landscape.

Integration with 3D Genome Sequencing Platforms

The resulting datasets align seamlessly with ChIA-PET, HiChIP, and Pore-C. Combined analyses link RNA–DNA contacts with DNA–DNA and protein–DNA interactions for a multi-layered 3D genomic model.

Applicable Across Species

Validated for plant tissues and adaptable to animal or microbial systems, the method accommodates diverse biological contexts in functional genomics, crop improvement, and molecular breeding research.

Applications of ChRD-PET

Promoter- and Enhancer-Centred RNA–DNA Interaction Mapping

ChRD-PET enables precise localisation of RNA–DNA contacts near active promoters and enhancers, helping researchers uncover how chromatin-associated RNAs influence transcription initiation and enhancer looping. This is essential for studying gene activation networks in plants and other eukaryotes.

Epigenetic Regulation and Histone Mark Integration

By combining chromatin immunoprecipitation with sequencing, ChRD-PET reveals how RNAs interact with histone-modified regions such as H3K4me3 or H3K27ac. These data clarify how epigenetic modifications and RNA molecules cooperatively regulate gene accessibility.

R-Loop and Noncoding RNA Analysis

The method profiles RNA–DNA hybrids derived from R-loops and classifies noncoding RNAs—including lncRNAs, snoRNAs and miRNAs—that anchor chromatin interactions. This insight supports studies on RNA-driven genome stability and chromatin organisation.

3D Genome Topology and Chromatin Loop Modelling

Integrated with 3D genome sequencing platforms such as ChIA-PET, HiChIP, and Pore-C, ChRD-PET provides promoter-to-enhancer interaction maps that connect RNA–DNA signals with chromatin loops and domains.

Functional Genomics and Crop Improvement Research

ChRD-PET supports comparative transcriptome studies, revealing how regulatory RNAs shape gene networks across developmental stages or environmental conditions. In agriculture, it offers tools to explore RNA-mediated chromatin regulation underlying stress response and yield traits.

Bioinformatics

Deliverables

• Raw Data: FASTQ files and QC reports.
• Processed Data: BEDPE tables, promoter-interaction matrices, and annotated hybrid lists.
• Visual Outputs: Contact heatmaps, RNA–DNA loop diagrams, and expression-linked scatterplots.
• Summary Report: Interactive HTML/PDF report including metrics, mapping statistics, and key visualisations ready for publication.

This comprehensive workflow enables multi-layered insights into how RNAs shape genome organisation and gene expression, giving researchers an integrative view of chromatin structure and function.

Sample Requirements

Shipping & storage guidelines:

• Freeze tissues or cell pellets in liquid nitrogen and ship on dry ice.
• Use rigid 2,000 mL foam box for tissue/cell shipments; cushion tubes to avoid breakage.
• Label tubes clearly (e.g., max 4 alphanumeric characters).
• For RNA, use RNase-free water or 10 mM Tris pH 8.0; ship on dry ice or with ice packs.

Follow regulatory and biosafety shipping guidelines (appropriate carrier e.g., FedEx, DHL).

QC thresholds employed by CD Genomics:

• Unique paired-end tags (RNA–DNA) ≥ 10 million per replicate.
• Mapping rate ≥ 70% to the reference genome.
• Duplicate rate ≤ 15%.
• For promoter-enhancer focus: cis/trans ratio consistent with published ChRD-PET data (local ~35.9 %, proximal ~8.8 %, distal ~55.3%)

FAQs

Pore-C Service

Long Read Sequencing for Chromatin Conformation

HiChIP Sequencing