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
| Tier | Analysis Modules | Description |
| Basic Analysis | Read Quality & Pre-processing | Adapter trimming, quality filtering, removal of low-confidence reads; QC report. |
| Alignment to Reference Genome | Map paired RNA–DNA tags to genome; output aligned BAM files and mapping statistics. | |
| Interaction Pair Assignment | Identify valid RNA–DNA tag pairs, classify into cis, proximal, distal contacts. | |
| Peak Detection & Annotation | Detect enriched DNA regions (promoters/enhancers), annotate interacting RNAs with gene context. | |
| Advanced Analysis | R-Loop & Non-coding RNA Characterisation | Integrate R-loop data, classify lncRNAs/snoRNAs/miRNAs engaging chromatin; deliver hybrid lists. |
| 3D Genome Integration | Combine with loop data from ChIA-PET, HiChIP or Pore-C; visualise RNA–DNA in chromatin topology. | |
| Interaction Network & Quantification | Build network models of RNA–DNA contacts, quantify interaction frequencies, generate heatmaps. | |
| Visual Reporting & Browser Tracks | Provide BEDPE tables, IGV/UCSC browser tracks, circular genome plots and publication-ready figures. |
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
| Sample Type | Recommended Input | Minimum Acceptable | Quality Metrics (Pre-QC) |
| Fresh plant tissue / cell culture | ≥ 10 million cells or ~50 mg fresh tissue | ≥ 5 million cells or ~25 mg tissue | OD₆₀₀/₄₈₀ for intact cells; minimal debris |
| Tissue with histone-antibody target (e.g., H3K4me3) | ≥ 100 µg chromatin equivalent | ≥ 50 µg chromatin equivalent | Chromatin fragment size ~200–500 bp after sonication |
| Crosslinked samples | Standard 1% formaldehyde ×10 min | Use ≤ 1% ×15 min if plant cell wall thick | No excessive cross-linking (DNA smear <500 bp) |
| RNA co-isolated sample (for R-loop or RNA mapping) | ≥ 1 µg total RNA, RIN ≥ 7 | ≥ 500 ng RNA, RIN ≥ 6 | A260/280 = 1.8–2.2; A260/230 ≥ 2.0 |
| Antibody-validated target (ChIP enrichment) | Verified antibody + pre-test pull-down | Dependent on pre-test result | Enrichment fold ≥ 10× vs input |
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
Q1: What is ChRD-PET and how does it differ from standard chromatin conformation methods?
ChRD-PET (chromatin-associated RNA–DNA interactions followed by paired-end-tag sequencing) specifically maps contacts between RNAs and DNA in the context of chromatin, unlike classical 3D genome methods (such as Hi-C) that only detect DNA–DNA contacts; by combining ChIP-based enrichment, RNA-DNA linker ligation and paired-end sequencing, it reveals the RNA dimension of genome architecture.
Q2: Which sample types and research applications are suitable for ChRD-PET?
ChRD-PET is suitable for fresh or frozen cell and tissue samples (plants, animals or microbes) where chromatin-associated RNAs are implicated. It is particularly effective for research on promoter/enhancer interactions, R-loops, noncoding RNAs, transcription-chromatin coupling and 3D genome topology in functional genomics, crop breeding and drug development.
Q3: What key data outputs can I expect from a ChRD-PET service?
Deliverables typically include raw FASTQ files, processed BEDPE interaction tables, interaction contact matrices, annotated RNA-DNA hybrid lists, browser tracks for IGV/UCSC, circular genome interaction plots and a summary report with metrics, all enabling downstream functional and structural analysis.
Q4: Can I integrate ChRD-PET data with other 3D genome platforms like ChIA-PET or HiChIP?
Yes. ChRD-PET is fully compatible with platforms such as ChIA-PET, HiChIP and Pore-C. Integration enables multi-layered analysis of RNA–DNA contacts, protein-DNA or DNA–DNA loops, and chromatin domain structures, thereby providing a holistic 3D genomic interaction landscape.
Q5: What are the quality controls and thresholds typical for ChRD-PET?
Quality control metrics include: mapping rate to the reference genome (typically ≥ 70 %), unique paired-end tags counts, duplication rate (low duplicates preferred), and cis-/trans-contact ratios reflecting local vs distal interactions (e.g., ~35-40 % local, ~50-60 % distal as seen in published datasets).
Reference
- Xiao, Q., Huang, X., Zhang, Y. et al. The landscape of promoter-centred RNA–DNA interactions in rice. Nat. Plants 8, 157–170 (2022).
For research purposes only, not intended for personal diagnosis, clinical testing, or health assessment