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iMARGI Sequencing Service: Genome-Wide RNA-Chromatin Interaction Mapping

Reveal the "dark matter" of the genome with our comprehensive iMARGI sequencing service. We map genome-wide interactions between chromatin-associated RNAs (caRNAs) and genomic DNA in situ, preserving native nuclear architecture. Get high-confidence, reproducible maps of lncRNA binding sites and co-transcriptional networks with our optimized CRO workflow. RUO.

  • Unbiased, genome-wide detection of RNA-DNA contacts
  • Differentiates cis (local) vs. trans (distal) interactions
  • Optimized for both coding (mRNA) and non-coding (lncRNA) transcripts
  • Deliverables: Valid interaction pairs, contact matrices, and visualization tracks
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3D illustration of iMARGI method showing RNA binding to genomic DNA in a cell nucleus.

Overview: Uncovering the Regulatory Role of RNA on Chromatin

Our iMARGI Sequencing Service (in situ Method for Analysis of RNA Genomic Interactions) offers a comprehensive, unbiased approach to mapping the "dark matter" of the nucleus: the complex network of interactions between chromatin-associated RNAs (caRNAs) and genomic DNA. While Hi-C sequencing has revolutionized our understanding of DNA-DNA contacts, it often overlooks the critical structural and regulatory role of RNA. iMARGI fills this gap by capturing RNA-chromatin interactions in their native nuclear context, preserving the spatial organization of the 3D genome.

Operated on a Research Use Only (RUO) basis, our service utilizes an optimized in situ chemistry that ligates RNA directly to spatially proximal DNA via a specialized linker. Unlike targeted methods (e.g., ChIRP-Seq) that require prior knowledge of the RNA target, iMARGI is a "one-to-all" and "all-to-all" discovery tool. It simultaneously reveals the genomic binding sites of thousands of transcripts—including long non-coding RNAs (lncRNAs), nascent mRNAs, and retrotransposons—providing a high-resolution map of the "RNome-Genome" interface.

Service Snapshot

  • Unbiased Discovery: No probes required; map all RNA-DNA interactions globally without prior hypothesis.
  • In Situ Preservation: Performed in intact nuclei to minimize non-specific interactions and background noise common in lysate-based methods.
  • Bimodal Detection: Distinguish between cis (nascent RNA at synthesis site) and trans (regulatory RNA at distal site) interactions.
  • Comprehensive Deliverables: From raw FASTQ to interaction matrices and Circos plots.

Service Highlights

Global "All-to-All" Mapping

Most RNA-chromatin methods (like ChIRP or RAP) are "one-to-all," requiring you to pick a single RNA target beforehand. iMARGI is "all-to-all," capturing the chromatin binding sites of every expressed RNA in the cell simultaneously. This is ideal for discovering novel regulatory lncRNAs or mapping the global impact of a perturbation.

In Situ Linker Ligation

We utilize a specialized bivalent linker that bridges the RNA 3'-end and the DNA 5'-end within the intact nucleus. This "proximity ligation" chemistry ensures that only physically interacting molecules are captured, significantly reducing the false positives associated with diffusion in solution-based protocols.

Distinguishing Cis vs. Trans

By mapping paired-end reads where one end is RNA and the other is DNA, we can computationally determine the genomic distance between the RNA's transcription start site and its chromatin binding site. This allows us to separate nascent transcripts (interacting in cis) from functional regulatory RNAs (interacting in trans).

Full-Stack Bioinformatics

Our service includes a rigorous bioinformatics pipeline. We don't just give you reads; we provide filtered interaction lists, contact matrices compatible with Hi-C visualization tools, and statistical significance scoring to separate biological signal from random collision noise.

Technical Comparison: iMARGI vs. GRID-seq vs. RADICL-seq

Feature iMARGI GRID-seq RADICL-seq ChIRP/RAP
Scope Global (All-to-All) Global (All-to-All) Global (All-to-All) Targeted (One-to-All)
Ligation Strategy Linker-mediated (RNA end to DNA end) Proximity Ligation (Linker) Proximity Ligation (Bridge) Biotin Probe Hybridization
Nascent RNA Capture High (Captures 3' end of nascent RNA) Moderate Moderate Variable
Distinguish Cis/Trans Yes (Bioinformatics definition) Yes Yes Yes
Input Requirement ~2-5 million cells ~1-5 million cells ~1-5 million cells >10-20 million cells
Bias Minimal sequence bias Restriction enzyme bias (MmeI) RNase digestion bias Probe design bias
Key Advantage High sensitivity for nascent transcripts & versatile application High specificity due to bivalent linker No restriction enzyme needed for RNA fragmentation High depth for single known target

Note: iMARGI's use of a specialized linker allows for the simultaneous sequencing of the RNA end and the DNA end in a paired-end read, ensuring that the "interaction" is physically linked before library preparation.

Our iMARGI Workflow: From Nuclei to Interaction Maps

Our end-to-end CRO workflow follows rigorous QC standards to ensure high-complexity libraries and reproducible contact maps.

1. Cell Crosslinking & Permeabilization

Cells are crosslinked with formaldehyde to covalently freeze RNA-protein-DNA complexes in situ. Nuclei are permeabilized to allow enzyme entry while maintaining nuclear integrity, preventing RNA leakage and false trans contacts. QC Check: Nuclei integrity inspection.

2. Chromatin Digestion & Linker Ligation

Chromatin is digested with a restriction enzyme (typically AluI or Csp6I) to expose DNA ends. A specialized linker—biotinylated and designed to ligate RNA 3'-OH ends to DNA 5'-phosphate ends—is introduced. This step physically bridges the interacting RNA and DNA molecules.

3. Reverse Transcription & Circularization

The ligated RNA is reverse transcribed into cDNA. The resulting chimera (cDNA-Linker-DNA) is circularized. This circularization brings the cDNA sequence and the genomic DNA sequence adjacent to each other, creating a verifiable junction.

4. Library Preparation & Sequencing

The circular molecules are linearized, enriched for biotin (selecting only valid RNA-DNA contacts), and PCR-amplified. We perform high-depth Paired-End sequencing (PE150) on NovaSeq platforms. Read 1 captures the RNA sequence, and Read 2 captures the interacting genomic DNA sequence.

5. Bioinformatics Analysis

Mapping reads to reference genome (RNA/DNA separately), Filtering PCR duplicates and rRNA reads, Interaction Calling (cis/trans classification), and Peak Calling for significant caRNA-DNA contacts.

Step-by-step iMARGI sequencing service workflow from crosslinking to data analysis.

Key Applications: From lncRNA Function to Transcriptional Regulation

The interplay between RNA and chromatin is fundamental to gene regulation. Our iMARGI service enables researchers to dissect these mechanisms with genomic precision.

Mapping lncRNA & caRNA Chromatin Occupancy

Long non-coding RNAs (lncRNAs) often act as scaffolds or guides for chromatin-modifying complexes. iMARGI identifies the specific genomic loci where these RNAs bind (*trans*-interactions). This is critical for characterizing the function of novel lncRNAs (e.g., *MALAT1*, *NEAT1*, *Xist*), determining whether they regulate genes in *cis* (neighboring) or *trans* (distal chromosomes), and identifying their potential roles in enhancer-promoter looping.

Characterizing Co-Transcriptional Processing

Because iMARGI captures nascent RNA still attached to the polymerase complex, it provides a unique window into co-transcriptional events. We can map nascent transcripts to visualize the immediate chromatin environment and analyze how splicing intermediates interact with chromatin.

Investigating 3D Genome Organization & Phase Separation

RNAs are key drivers of nuclear compartmentalization and phase separation (e.g., nucleolus, nuclear speckles). By integrating iMARGI data with Hi-C maps, researchers can correlate caRNA abundance with Topologically Associating Domain (TAD) boundaries and identify RNAs that bridge distinct chromosomal territories.

Viral RNA-Host Interaction

For infected cells, iMARGI can map where viral RNAs interact with the host genome, shedding light on how viruses hijack host transcriptional machinery or integrate into the genome.

Why Choose CD Genomics for iMARGI?

Pioneer in 3D Genomics

We offer one of the most comprehensive portfolios of 3D genomics services, including Hi-C, Micro-C, and ChIA-PET, allowing for seamless multi-omics integration.

Optimized Chemistry

Our protocol includes specific improvements to reduce background from ribosomal RNA (rRNA) and non-specific ligation, maximizing the yield of informative unique mapped pairs.

Data You Can Trust

We provide extensive QC reports, including "interaction composition" metrics (cis/trans ratio, genic/intergenic distribution) that allow you to verify library quality before diving into analysis.

Customizable Bioinformatics

Beyond standard mapping, we offer custom analyses like motif enrichment at binding sites, differential interaction analysis between conditions, and integration with ChIP-seq data.

Sample Requirements

Sample Type Recommended Input Storage/Transport Notes
Cell Lines 5 x 106 viable cells Cryopreserved (DMSO) or Flash Frozen pellet Viability >90% required. Rinse with PBS before freezing.
Fresh Tissue >50 mg Flash Frozen or RNAlater Tissue must be homogenized gently to isolate intact nuclei.
Blood/PBMC >5 mL whole blood EDTA tubes / Isolated PBMCs Isolate PBMCs immediately before freezing to ensure viability.
Crosslinked Cells 5 x 106 cells Fixed with 1% Formaldehyde Contact us for specific fixation protocols (critical step).

Key Deliverables

Raw Data & Matrix
FastQ files (Clean reads) and Interaction Matrix (.hic / .cool) compatible with Juicebox/HiGlass for visualization.

Circos Plots
Global visualization of inter-chromosomal interactions, highlighting "hub" RNAs that contact multiple genomic regions.

Genomic Tracks
BigWig files showing RNA tracks (gene body coverage) and DNA tracks (binding sites).

Interaction List & QC
BEDPE files listing significant RNA-DNA pairs with p-values, plus a detailed QC report on library complexity and cis/trans ratios.

Case Study: Interplay Between caRNA and 3D Genome Organization

Chromatin-associated RNAs (caRNAs) are integral components of the nuclear architecture, yet their specific contribution to 3D genome folding remains an area of active investigation. A 2023 study published in Nature Communications utilized iMARGI to systematically analyze the relationship between caRNA distribution and 3D genome structures (TADs and A/B compartments) in human cells.

The researchers performed iMARGI sequencing on K562 and H1 cells to map genome-wide RNA-chromatin interactions. These maps were integrated with Hi-C data to correlate caRNA accumulation with chromatin insulation scores and compartment status.

The iMARGI data revealed a striking enrichment of caRNAs at TAD boundaries. Specifically, high levels of caRNA-chromatin interactions were predictive of strong TAD insulation. Distinct classes of RNAs were found to segregate into A (active) and B (inactive) compartments, suggesting that RNAs help maintain the biophysical separation of these chromatin states via phase separation mechanisms.

Heatmaps showing chromatin-associated RNA interactions mapped by iMARGI.

This study demonstrated that iMARGI is a powerful tool for linking the "RNome" to the "3D Genome," providing evidence that caRNAs are not just passive products of transcription but active architects of chromatin organization.

(Source: Adapted from Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization, Nat Commun 2023)

Demo Results (Representative Examples)

  • Interaction Matrix: A heatmap format compatible with standard viewers (Juicebox, HiGlass).
  • Circos Plots: Global visualization of inter-chromosomal interactions, highlighting "hub" RNAs.
  • Genomic Tracks: BigWig files showing RNA tracks (gene body coverage) and DNA tracks (binding sites).
  • Interaction List: A tabular file listing every significant RNA-DNA pair with statistical confidence scores.

iMARGI sequencing data showing RNA-DNA contact matrix.Contact Matrix

Circos plot visualization of global RNA-chromatin interactions.Circos Plot

Genomic tracks showing RNA and DNA mapping from iMARGI.Genomic Tracks

Frequently Asked Questions

References

  1. Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization in human cells. Nature Communications. 2023.
  2. RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions. Nature Communications. 2020.
  3. Genome-Wide Technologies to Study RNA–Chromatin Interactions. Non-Coding RNA. 2020.
  4. Regulation of nuclear transcription by mitochondrial RNA in endothelial cells. eLife. 2023.
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High-confidence 3D genomics services for chromatin interaction analysis and regulatory insight.

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