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Circular Chromosome Conformation Capture Sequencing (4C-seq): A Targeted Solution for Chromatin Interaction Analysis

Accelerate your discovery of regulatory loops, enhancer-promoter contacts, and 3D genome structure with our targeted 4C-seq service — optimized for low-input samples, cost efficiency, and reliable, publication-quality data.

  • Focused locus-centric mapping — pinpoint chromatin contacts for any gene or regulatory region of interest.
  • Flexibility & scalability — supports single-viewpoint, multiplex, long-read, low-input, and capture-enhanced workflows to match project size and sample availability.
  • End-to-end service — we handle sample prep, primer design, sequencing, and bioinformatics — delivering QC-ready results you can trust.
  • High reproducibility & regulatory-grade QC — ideal for drug discovery, functional genomics, or translational research.
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4C-seq chromatin interaction map showing enhancer-promoter loops, highlighting precision, flexibility, and cost-efficiency for targeted regulatory studies.

What Is 4C-seq? — Targeted Chromatin Interaction Profiling

Circular Chromosome Conformation Capture Sequencing (4C-seq) is a targeted genomic assay designed to map all chromatin interactions connected to a specific locus of interest. Rather than producing broad, genome-wide contact maps, 4C-seq delivers high-resolution, locus-centric profiles — ideal for investigating regulatory loops such as enhancer–promoter contacts or long-range regulatory elements.

Because 4C-seq focuses only on interactions involving the selected “bait” region, it requires comparatively modest sequencing depth and lower sample input. This makes it an efficient, cost-effective tool when working with limited material (e.g., rare cell types, primary tissues) or when you want to validate specific hypothesized contacts without the data overload of genome-wide methods.

In practice, 4C-seq is especially useful for:

  • Identifying enhancer–promoter loops and long-range regulatory interactions that control gene expression.
  • Validating candidate regulatory elements from epigenomic data, GWAS, or transcription-factor binding studies.
  • Working with low-input or precious samples while still obtaining high-quality, publication-ready data.

Why Choose 4C-seq?

  • Precision: Focuses on specific loci of interest, offering highly reliable data.
  • Cost-Effective: Compared to genome-wide approaches, 4C-seq is more affordable, with lower sequencing depth and sample input requirements.
  • Low-Input Compatibility: Works efficiently with limited sample sizes, such as rare cells or archival tissue, making it ideal for challenging samples.
  • Flexibility: Choose between single-locus validation or multiplex assays to match your research scale.

4C-seq vs. Hi-C

Scale One-to-all (single bait) All-to-all (genome-wide)
Sequencing Depth Low to moderate (~1–2 million reads) High (hundreds of millions, often >100M reads)
Input Requirement Low (suitable for small samples, primary cells) High (large cell numbers or tissue)
Data Complexity Manageable; focused on relevant locus Very large; requires heavy computational resources
Ideal For Validating specific regulatory interactions, enhancer-promoter loops, locus-centric studies Global chromatin architecture, TAD discovery, genome-wide 3D structure

4C-seq Service Modules — Customizable for Your Needs

To support diverse research needs, we offer the following 4C-seq modules: These modular options allow you to tailor the 4C-seq service to your project’s size, complexity, and objectives — from simple locus validation to in-depth chromatin architecture exploration.

Module 1: Standard 4C-seq

Core assay for one-to-all chromatin interaction profiling around a single bait locus. Ideal for enhancer–promoter validation or regulatory region studies.

Module 2: Multiplex 4C-seq (Multi-Viewpoint)

Enables simultaneous interrogation of several loci within the same library. Great for comparative studies, screening multiple regulatory sites, or building regulatory maps.

Module 3: Inverse PCR-Optimized 4C

Enhanced inverse PCR protocol to improve sensitivity and specificity in complex genomic regions. Recommended for repeat-rich or GC-rich contexts.

Module 4: Long-Read 4C Enhancement (MC-4C, Nanopore)

Combines 4C with long-read sequencing (e.g., Nanopore) to capture long-range contacts, structural variants, or multi-fragment contacts missed by short-read sequencing.

Module 5: Low-Input 4C Workflow

Optimized for limited or precious samples (e.g., primary cells, rare tissues). Refined lysis, digestion, and purification steps maximize yield and data quality from small inputs.

Module 6: Capture-Enhanced 4C Option

Uses hybridization-based capture enrichment to focus on predefined genomic regions, improving sensitivity and reducing background noise, useful for studying weak or rare interactions.

Module 7: 4C-qPCR / 4C-ddPCR Validation

Offers quantitative validation of chromatin interactions via qPCR or droplet digital PCR (ddPCR), providing an orthogonal layer of confidence for regulatory or therapeutic studies.

Overview of 4C-seq Workflow

1. Crosslinking
Cells or tissue samples are treated (e.g., with formaldehyde) to freeze chromatin in its native 3D conformation.

2. Primary Restriction Digest
Crosslinked chromatin is enzymatically cut into fragments using a restriction enzyme.

3. Ligation under Dilute Conditions
Fragments that were close in 3D space are ligated together, favouring intra-complex ligation.

4. Reverse Crosslink & Purification
Crosslinks are reversed, and DNA is purified.

5. Secondary Digest and Circularisation
Purified DNA is digested again (with a different restriction enzyme) and ligated at dilute concentrations to create circular DNA fragments. This ensures that interacting fragments are represented as circles accessible for inverse PCR.

6. Inverse PCR / Primer Amplification
Using primers designed around the “bait” fragment, circularized DNA is amplified to enrich interacting fragments of interest.

7. Library Preparation & Sequencing
Amplified products are prepped for next-generation sequencing (NGS), typically on Illumina platforms.

8. Data Analysis
Sequencing reads are mapped and processed; contact frequencies are calculated and visualized as interaction profiles across genomic coordinates. Specialized tools such as FourCSeq or 4C-ker are often used.

4C-seq service workflow

Applications of 4C-seq — Where This Service Delivers Value

Because of 4C-seq’s flexibility and depth, it is valuable across basic research, translational studies, and drug development pipelines. Our 4C-seq service supports a wide range of research applications:

Enhancer–Promoter Interaction Mapping

Validate distal regulatory elements controlling gene expression in development, disease, or cell differentiation.

Functional Genomics & Gene Regulation Studies

Dissect how chromatin loops and 3D architecture affect gene expression, alternative splicing, or epigenetic regulation.

Non-coding Variant Functionalization (GWAS follow-up)

Link disease-associated non-coding variants (SNPs) to their target genes via physical chromatin contacts.

Drug Discovery & Therapeutic Target Validation

Identify regulatory interactions relevant to gene targets, assess chromatin changes under drug treatment, or study drug-induced chromatin remodeling.

Structural Genomics & Chromatin Architecture Research

Explore long-range chromatin contacts, topologically associating domains (TADs), or structural rearrangements.

Low-Input or Rare Sample Studies

Apply to primary cells, patient-derived samples, sorted rare populations — scenarios where material is limited.

Why Choose CD Genomics for 4C-seq?

  • Comprehensive, End-to-End Service: From primer design to data analysis, we manage the full 4C-seq workflow. You receive raw data, processed files, and interpreted reports — ready for publication or regulatory use.
  • Modular & Customizable: Our seven service modules let you tailor the assay format to your project’s scale, input, and complexity.
  • Expertise in Complex or Low-Input Samples: We have extensive experience with challenging sample types (primary cells, tissues, archived samples), long-read capture, and multiplexed assays.
  • Robust Bioinformatics & Data Quality: We apply industry-standard tools (e.g., FourCSeq, 4C-ker), QC pipelines, and integrate epigenomic annotations to maximize biological value.
  • Transparency & Collaboration: Clear documentation, reproducible pipelines, and open communication — we collaborate with you to design the optimal 4C-seq assay for your research needs.

Data Analysis & Reporting — From Raw Reads to Biological Insights

Once sequencing is complete, our bioinformatics pipeline delivers: This end-to-end data analysis ensures you can interpret and act on chromatin interaction data rapidly, with clarity and confidence.

Read mapping and normalization
Sequencing reads are aligned; contact frequencies are normalized to account for restriction fragment biases and sequencing depth. Pipelines such as FourCSeq or 4C-ker are used.

Contact frequency profiling
Genome-wide profiles anchored on the bait locus, visualized as contact plots. Interaction peaks above background indicate likely loops or regulatory contacts.

Differential comparison (if multiple conditions)
Quantitative comparison between samples (e.g., treated vs control, cell types, developmental stages) to detect changes in chromatin interactions. Tools like 4C-ker allow statistical detection of differential contacts.

Custom annotation and integration
Overlay contacts with gene models, epigenomic data (ChIP-seq, ATAC-seq, histone marks) to interpret regulatory significance.

Publication-ready reports
We provide: Contact maps and plots (jpg, PDF); Raw and processed data (BAM, bedGraph, bigWig); Methodology summary, QC metrics, and recommendations for follow-up (e.g., qPCR validation, functional assays).

Case Study: Linking a Prostate-Cancer Risk Enhancer at 8q24 to Distant Targets Using 4C-seq

Source: Mingyang Cai et al., “4C-seq revealed long-range interactions of a functional enhancer at the 8q24 prostate cancer risk locus.” Scientific Reports, 2016.

Genetic studies have identified the 8q24 region as a hotspot for prostate cancer risk. However, risk variants in this locus locate to a gene-poor region, making it unclear how they contribute to disease. The proto-oncogene MYC lies ~200 kb away, but direct functional connections were not established. The study aimed to map genome-wide chromatin contacts of a functional enhancer (termed “AcP10”) inside the 8q24 risk region. The goal was to reveal potential target genes regulated through long-range chromatin looping, thus offering mechanistic insight into how non-coding risk variants may influence prostate cancer risk.

  • The authors performed 4C-seq in two prostate cancer cell lines: LNCaP (androgen-sensitive, lymph-node metastatic) and C4-2B (androgen-independent, bone-metastasis derived).
  • They used BglII as primary restriction enzyme; following cross-linking and ligation, inverse PCR primers were designed around the AcP10 enhancer as “bait.”
  • Two biological replicates per cell line were prepared, then sequenced on Illumina platforms.
  • The sequencing reads were processed using a custom analysis pipeline; contact profiles (cis and trans) were generated. Interactions reproducibility was confirmed across replicates via bin counts (2 Mb bins for trans, 1 Mb for cis). Circos plots were used to visualize genomic contacts.

The 4C-seq data revealed widespread long-range interactions between the 8q24 AcP10 enhancer and multiple genomic regions across the genome, including cis contacts over >300 kb and trans contacts on different chromosomes. Notably, interactions included the region containing MYC, supporting the hypothesis that the non-coding risk enhancer physically contacts a known oncogene. The reproducibility across replicates demonstrated robust chromatin interaction mapping. Circos plots (shown in Fig. 1A of the paper) illustrated the genome-wide network of contacts.

Genome-wide 4C-seq contact map

This study demonstrates that a non-coding enhancer within a risk locus can form stable, long-range chromatin loops reaching distant genes such as MYC. 4C-seq provided direct evidence linking genetic risk variants to functional regulatory interactions. Such data help bridge the gap between GWAS associations and mechanistic understanding of disease risk. This example illustrates how 4C-seq can reveal regulatory loops connecting non-coding risk loci to critical target genes — a powerful demonstration of the value of our 4C-seq service.

Demo Results

  • Processed count matrices linked to spatial coordinates and cluster labels.
  • Spatial maps: UMAP and t-SNE plots, along with cell cycle and trajectory visualizations.
  • Tables of marker genes, enriched functions, interaction metrics, and spatially variable genes.
  • A concise analysis report describing methods, key quality metrics, and biological findings.

4C-seq multiplex panel heatmap showing interaction strength between genomic fragments across multiple samples.Heatmap of Interaction Frequency

UMAP plot displaying chromatin interaction profiles, with samples clustered based on interaction patterns in 4C-seq data.UMAP or t-SNE Plot (Spatial Mapping)

Interaction bar plot displaying chromatin contact frequencies across different loci in a 4C-seq experiment.Interaction Bar Plot

Genome browser view showing 4C-seq contact data integrated with gene annotations for regulatory element mapping.Gene Annotation Integration (Contact Mapping with Genes)

Frequently Asked Questions (FAQ)

References

  1. Karasu, N., & Sexton, T. (2020). 4C-Seq: Interrogating Chromatin Looping with Circular Chromosome Conformation Capture. Methods in Molecular Biology (Vol. 2157, pp. 19–34). Springer.
  2. Krijger, P. H. L., Geeven, G., Bianchi, V., Hilvering, C. R. E., & de Laat, W. (2019). 4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis. Methods.
  3. Huang, Z., Wang, C., Treuter, E., & Fan, R. (2022). An optimized 4C-seq protocol based on cistrome and epigenome data in the mouse RAW264.7 macrophage cell line. STAR Protocols, 3(4).
  4. Marsman, J., & Colleagues. (2022). Circular Chromosome Conformation Capture Sequencing (4C-Seq) in Primary Adherent Cells. In Methods in Molecular Biology (Vol. 2458). Springer.
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