5C-seq Service

Chromosome Conformation Capture (3C) converts physical chromatin contacts into ligation products, but classic 3C typically quantifies interactions one-by-one by PCR. 5C (3C-Carbon Copy) extends 3C into a high-throughput format by using highly multiplexed ligation-mediated amplification (LMA) to "copy" and amplify a selected portion of a 3C library, followed by detection on microarrays or quantitative DNA sequencing (5C-seq). CD BioSciences' 5C-seq Service delivers sequencing-based 5C to profile chromatin interaction networks across large genomic regions—enabling efficient discovery and comparison of enhancer–promoter connectivity, regulatory neighborhoods, and regional chromatin folding at high resolution and high coverage.

Introduction to 5C-seq

In 5C-seq, a 3C library is generated first. A mixture of 5C forward and 5C reverse primers is then annealed to the 3C library and ligated in a multiplex reaction, such that only primer pairs positioned across a valid ligation junction are joined. The ligated primers are amplified using universal primer tails, producing a quantitative "carbon copy" of the targeted portion of the 3C library defined by your primer panel.

Primer architecture is a core feature of 5C: forward primers share a common T7-containing tail, reverse primers share a common T3c tail and are 5' phosphorylated to facilitate ligation; and because primers for a given restriction fragment are complementary, only one primer (forward or reverse) per fragment is used in a given design.

Fig.1 5C data correlations cluster by condition. (Beagan, J. A., et al., 2020)

Features of 5C-seq

1. Many-vs-many interaction profiling within selected genomic regions, enabling large-scale mapping beyond singleplex 3C.
2. Dense regional interaction matrices using alternating forward/reverse primer layouts to rapidly obtain a map-like view of regional folding.
3. Sequencing-based quantification (5C-seq) for scalable, high-dynamic-range readout without custom array manufacturing.
4. Ultra-high-resolution, targeted mapping on a subset of the genome, enabled by modern designs such as in situ 3C + double alternating primer schemes and standardized primer-design workflows (e.g., My5C).

Our Services

CD BioSciences provides an end-to-end 5C-seq service from region/primer-panel strategy through wet-lab library construction, sequencing, and analysis. We emphasize feasibility review (restriction layout, uniqueness, repeat avoidance), reproducible 3C/5C execution, and delivery of interpretation-ready interaction outputs aligned with your study goals.

Study Design & Region Strategy

1. Define target region(s) (typically hundreds of kb to multi-Mb) and desired output (anchor profiles vs matrix maps)
2. Plan experimental comparisons (replicates, conditions, time course, perturbations)

3C Template Generation

1. Crosslinking, restriction digestion, and proximity ligation to generate a 3C library (ligation products reflect in vivo contact frequencies)
2. QC checks for digestion/ligation performance prior to 5C conversion

5C Primer Panel Design & Validation Strategy

1. Primer panel designed for restriction fragments in your region(s) using standardized tools (e.g., My5C) and double-alternating schemes when appropriate
2. Uniqueness and repeat avoidance review to reduce over-amplification artifacts

Multiplex LMA to Generate the 5C Library

1. Anneal primers to the 3C library, ligate adjacent primer pairs (e.g., Taq ligase), and amplify via universal tails to generate the "carbon copy" 5C library
2. Library QC (expected amplicon patterns; cleanup)

Sequencing & Data Analysis

1. Sequencing-ready library prep and paired-end sequencing (depth scaled to panel size/complexity)
2. Panel-aware mapping and normalization; generation of interaction matrices/heatmaps and, where applicable, loop/enrichment summaries (project-dependent)

Supported Sample Types

1. Cultured Cells
2. Primary Cells
3. Fresh-Frozen Tissues

1. Time-Course or Drug-Treated Samples
2. Cell Differentiation or Induction Models
3. Other Types (Please Inquire)

Our Advantages

1. Targeted, High-Resolution 3D Mapping at Scale: 5C uses multiplex LMA to enable large-scale interaction profiling within defined genomic regions, extending beyond single-interaction 3C assays.
2. Flexible Design for Different Biological Questions: Supports both fixed-element interaction profiling and dense regional mapping via alternating primer strategies.
3. Primer Design Discipline for Cleaner Data: Repeat-recognizing primers can produce excessive ligation products; we enforce repeat avoidance and feasibility checks as a core quality step.
4. Modern 5C-seq Improvements Available: In situ 3C + double alternating primer designs (5C-ID–style) have been reported to improve map quality/resolution and reduce input needs relative to canonical 5C.

Build high-resolution chromatin interaction maps across your genomic regions of interest with CD BioSciences' 5C-seq service—contact us to discuss your target loci, preferred design mode (anchor profiles vs regional matrices), sample type, and experimental comparisons.

Reference

1. Beagan, J. A., et al. (2020). Three-dimensional genome restructuring across timescales of activity-induced neuronal gene expression. Nature neuroscience, 23(6), 707–717.