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Micro-C Service: Nucleosome-Resolution 3D Genome Mapping

Go beyond the limits of restriction enzymes. Our Micro-C Service utilizes Micrococcal Nuclease (MNase) digestion to fragment chromatin down to single nucleosomes, enabling nucleosome-resolution 3D genome mapping. Capture fine-scale enhancer-promoter loops (< 20kb) and precise domain boundaries that standard Hi-C misses, providing the ultimate structural clarity for your regulatory genomics research (RUO).

  • Nucleosome Resolution: Map interactions at ~150bp precision (vs. ~5kb for Hi-C).
  • No Enzyme Bias: MNase digestion eliminates restriction site limitations.
  • Enhanced Loop Detection: Visualize distinct "dots" and "stripes" for clearer V2G assignment.
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Micro-C workflow showing MNase digestion and nucleosome-level fragmentation

Overview: Breaking the Resolution Barrier of Standard Hi-C

In the field of 3D genomics, resolution is the defining constraint. Standard Hi-C (High-Throughput Chromosome Conformation Capture) has been the workhorse for mapping Topological Associating Domains (TADs) and compartments. However, Hi-C relies on restriction enzymes (REs) such as HindIII or DpnII to fragment chromatin. This introduces an inherent "resolution floor": the distribution of restriction sites is non-uniform, leaving "blind spots" in AT-rich regions or restriction-poor loci. Furthermore, the average fragment size (typically 400bp–4kb) limits the ability to resolve interactions smaller than 5–10 kb, obscuring the fine-scale regulatory loops that drive gene expression.

Micro-C (Micrococcal Nuclease Hi-C) shatters this barrier. Instead of restriction enzymes, Micro-C utilizes Micrococcal Nuclease (MNase) to digest chromatin. MNase cuts exposed linker DNA between nucleosomes, generating a library of mononucleosomes (~150 bp) and dinucleosomes.

Our Micro-C Service leverages this enzymatic advantage to map chromatin architecture at nucleosome resolution. By bypassing restriction site bias and reducing fragment size to the biological limit, Micro-C achieves an unprecedented signal-to-noise ratio. This allows for the visualization of fine-scale chromatin features—such as enhancer-promoter loops (< 20 kb), architectural stripes, and precise loop anchors—that are often blurred or invisible in standard Hi-C maps.

(Note: This service is for Research Use Only. It is not intended for use in diagnostic procedures or clinical decision-making.)

Key Technical Advantages

  • Nucleosome Precision: Map interactions at ~150–200 bp resolution, compared to 1–5 kb for high-depth Hi-C.
  • No Enzyme Bias: MNase digests uniformly across the genome, eliminating coverage gaps caused by restriction motif distribution.
  • High Signal-to-Noise: Superior detection of short-range interactions, clearing the "diagonal" to reveal proximal enhancer-promoter contacts.

Applications: Seeing the Unseen in Chromatin Architecture

Micro-C is not simply "Hi-C with more reads"; it is a structural probe for the fine-scale organization of the genome. It is the preferred method for researchers investigating gene regulation mechanisms, structural variation, and architectural proteins.

Precise Enhancer-Promoter Loop Calling (V2G)

A major challenge in Hi-C analysis is "Variant-to-Gene" (V2G) assignment. Many functional enhancer-promoter loops span short genomic distances (< 50 kb). Because Micro-C fragments are nucleosome-sized, the "diagonal" is much tighter, unmasking short-range loops and allowing definitive assignment of non-coding variants to target promoters.

Resolving Fine-Scale TAD Boundaries & Stripes

While Hi-C identifies TADs as broad triangles, Micro-C reveals internal substructures. It sharply resolves "stripes" (or flames) extending from TAD boundaries, representing Cohesin loop extrusion, and visualizes Sub-TADs nested within larger domains.

Determining Nucleosome Positioning & Phasing

Unlike any other 3C-based method, Micro-C retains 1D nucleosome positioning information. By analyzing read pileup at anchors, you can determine precise nucleosome phasing around CTCF binding sites or TSSs, integrating 1D occupancy with 3D topology.

Transcription-Coupled Folding

Micro-C is sensitive enough to detect "transcriptional bursts" and the formation of Enhancer-Promoter (E-P) and Promoter-Promoter (P-P) loops associated with RNA Polymerase II pausing and elongation.

Our Micro-C Workflow: MNase Digestion Protocol

The Micro-C protocol is technically demanding, requiring precise enzymatic titration to ensure optimal chromatin fragmentation without over-digestion.

Step 1: Dual Cross-linking (Optional but Recommended)
We often employ Dual Cross-linking (DSG + FA). Disuccinimidyl glutarate (DSG) stabilizes protein-protein interactions, creating a robust chromatin network that preserves "long-range" loops during aggressive MNase digestion.

Step 2: Chromatin Fragmentation via MNase
Nuclei are permeabilized and treated with Micrococcal Nuclease (MNase). Unlike restriction enzymes, MNase cuts exposed linker DNA between nucleosomes. QC Checkpoint: We verify a fragmentation profile dominated by mononucleosomes (~150 bp) and dinucleosomes (~300 bp) via electrophoresis.

Step 3: End Repair & Biotinylation
Digested DNA ends are repaired, and a biotinylated nucleotide is incorporated to mark the ligation junctions.

Step 4: Proximity Ligation
Under dilute conditions, DNA ends held together by protein complexes are ligated. Since fragments are nucleosome-sized, this captures true physical proximity.

Step 5: Library Generation & Deep Sequencing
Cross-links are reversed, and biotinylated junctions enriched. The library is sequenced on Illumina NovaSeq. We recommend Deep Sequencing (600M – 1 Billion reads) to fully realize resolution benefits.

Micro-C workflow diagram showing MNase digestion and nucleosome-level fragmentation

Demo Results: The "Sharpness" of Micro-C

Visualizing Micro-C data reveals a striking improvement in clarity compared to traditional methods.

Figure 1: Genomic Track Comparison (200kb Window)

Left Panel (Standard Hi-C): The interaction matrix appears pixelated or "blocky." While the main TAD triangle is visible, the internal structure is a uniform haze. Short-range interactions near the diagonal are obscured.

Right Panel (Micro-C): The same region appears in high definition.

  • Dots: Distinct focal enrichments (loops) appear clearly off-diagonal, connecting specific enhancer and promoter loci.
  • Stripes/Flames: Fine vertical or horizontal lines extend from TAD boundaries, visualizing Cohesin loop extrusion.
  • Diagonal: The main diagonal is tight and sharp, allowing detection of proximal interactions (< 5 kb).

Comparison of chromatin interaction resolution between Standard Hi-C and Micro-C sequencingFigure 1: Resolution Revolution

Comparison: Micro-C vs. Standard Hi-C vs. Capture-C

Feature Micro-C Standard Hi-C Capture-C / Capture Hi-C
Digestion Enzyme MNase (Exonuclease/Endonuclease) Restriction Enzyme (e.g., DpnII, HindIII) Restriction Enzyme
Resolution Limit Nucleosome (~150 bp) Restriction Fragment (~4 kb) / Bin (~5 kb) Fragment Level (Targeted)
Sequence Coverage Uniform (No blind spots) Biased by RE motif distribution Targeted Regions Only
Loop Detection < 20 kb loops & long-range > 20-50 kb loops (Short-range is noisy) Targeted loops (High sensitivity)
Key Output Fine-scale topology, Stripes, Nucleosome phasing TADs, Compartments A/B, Large Loops Promoter interactomes (One-to-All)
Cost / Depth High (>800M reads) Moderate (300M - 600M reads) Low to Moderate (Targeted)

Choose Standard Hi-C for global compartment analysis. Choose Capture Hi-C for specific promoter interactions. Choose Micro-C if you need the highest possible resolution genome-wide.

Case Study: Ultrastructural Details of Chromosomes (Mol Cell 2020)

The following case study highlights the capabilities of Micro-C in mammalian systems, based on the foundational work by Krietenstein et al.

The Challenge

Researchers suspected that standard Hi-C was missing a significant portion of chromatin loops due to resolution limits. While Hi-C identified ~10,000 loops in mouse embryonic stem cells (mESCs), functional data suggested many more regulatory contacts existed.

The Solution

The team applied Micro-C with dual cross-linking (DSG+FA) to mESCs and sequenced to high depth (~1 billion unique contacts). They compared this dataset to high-depth HindIII Hi-C.

The Results

Micro-C identified approximately 20,000 additional loops that were invisible in Hi-C maps. Most were short-range (< 100 kb) enhancer-promoter interactions. The study also provided the first clear genome-wide visualization of "stripes" originating from CTCF sites.

Micro-C data showing fine-scale enhancer-promoter loops invisible to Hi-C

The Conclusion

Micro-C provides an ultrastructural view of the genome, bridging the gap between nucleosome positioning and large-scale folding.

Source: Krietenstein, N., et al. "Ultrastructural Details of Mammalian Chromosome Architecture." Molecular Cell (2020).

FAQ: Input & Sequencing Depth

References

  1. Hsieh, T.H., et al. Mapping Nucleosome Resolution Chromosome Folding in Yeast by Micro-C. Science. 2015;350(6263):931-940.
  2. Krietenstein, N., et al. Ultrastructural Details of Mammalian Chromosome Architecture. Molecular Cell. 2020;78(3):554-573.
  3. Hsieh, T.H., et al. Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding. Molecular Cell. 2020;80(5):845-861.
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