Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful next-generation method for genome-wide mapping of histone post-translational modifications (PTMs), transcription factors, and chromatin regulators. This guide details the optimized CUT&RUN workflow, from experimental design to data analysis.
What is CUT&RUN?
CUT&RUN represents a significant advancement over traditional chromatin immunoprecipitation sequencing (ChIP-seq). Developed by Dr. Steven Henikoff's group, this technique builds on Chromatin ImmunoCleavage (ChIC) principles where a protein A/G-micrococcal nuclease (pAG-MNase) fusion protein is recruited to antibody-bound chromatin targets for precise in situ cleavage.
The key advantages of CUT&RUN include:
- Dramatically reduced background signal
- Compatibility with low cell inputs (as few as 5,000 cells)
- Elimination of cross-linking and chromatin shearing steps
- Higher resolution mapping with minimal artifacts
- Reduced sequencing requirements (3-8 million reads per sample)
Key Components of the CUT&RUN Workflow
Essential Reagents and Equipment
Successful CUT&RUN experiments require specific reagents designed for optimal performance:
- pAG-MNase: The essential enzyme fusion that cleaves antibody-bound chromatin
- Concanavalin A (ConA) magnetic beads: For cell immobilization
- Validated antibodies: Critical for target specificity
- Digitonin-containing buffers: For cell permeabilization
- Specialized magnetic racks: For efficient sample processing
Proper equipment selection significantly impacts reproducibility. An 8-strip PCR tube format with compatible magnetic racks enables high-throughput processing and consistent results.
Experimental Design Considerations
Careful planning is crucial for successful CUT&RUN experiments. Key considerations include:
Control Reactions:
- Negative control (IgG antibody)
- Positive control (H3K4me3 for PTMs, CTCF for transcription factors)
- Spike-in controls for normalization and quality assessment
Cell Input Optimization:
While the protocol works with 500,000 cells, successful results have been obtained with as few as 5,000 cells for abundant targets. The optimal cell number should be determined empirically for each target and cell type.
Digitonin Titration:
Cell permeabilization requires optimization for each cell type. Perform digitonin titrations (0.001%-0.1%) to identify the minimum concentration that achieves >95% permeabilization without causing cell lysis.
Step-by-Step Protocol Overview
Day 1: Sample Preparation and Antibody Binding
ConA Bead Activation (30 minutes)
Activate ConA beads in specially formulated buffer to prepare for cell binding. Batch processing all beads needed for an experiment ensures homogeneity across reactions.
Cell Binding to Activated Beads (30 minutes)
Harvest cells and immobilize them onto ConA beads. The protocol supports various sample types including suspension cells, adherent cells, nuclei, and cryopreserved material. Quality control checks at this stage are essential for success.
Antibody Binding (Overnight)
Incubate samples with target-specific antibodies in digitonin-containing buffer. Antibody selection is critical—not all ChIP-validated antibodies work well in CUT&RUN. 👉 Explore validated antibody options
Day 2: Chromatin Cleavage and Library Preparation
pAG-MNase Binding (30 minutes)
Add pAG-MNase to antibody-labeled chromatin. Gentle handling is crucial at this stage as samples may become clumpy.
Targeted Chromatin Digestion (2 hours)
Activate MNase with calcium to cleave antibody-bound chromatin. The cleaved fragments diffuse into the supernatant for collection.
DNA Purification and Quality Assessment
Purify released DNA fragments using specialized kits designed to capture fragments as small as 50 bp. Quantify yields but avoid size distribution analysis at this stage—proceed directly to library preparation.
Library Preparation and Sequencing
Prepare sequencing libraries using optimized kits specifically designed for CUT&RUN. Only 3-8 million paired-end reads are typically needed per sample, enabling extensive multiplexing.
Quality Control and Troubleshooting
Implementing robust quality control measures throughout the protocol is essential for success:
Sample Integrity Checks:
- Verify cell viability and absence of clumping before bead binding
- Confirm successful ConA bead conjugation using Trypan blue exclusion
- Monitor permeabilization efficiency
Experimental Success Indicators:
- Higher DNA yields in positive controls compared to IgG negative controls
- ~300 bp peak in library fragment size distribution (mononucleosome + adapters)
- Expected genomic distribution patterns in sequencing data
Avoid traditional ChIP-seq quality control methods like pre-library fragment analysis or qPCR validation, as these are not appropriate for CUT&RUN and may lead to incorrect conclusions.
Advanced Applications and Modifications
Special Sample Types
The protocol can be adapted for various challenging sample types:
Adherent Cells:
Use mild trypsin treatment (0.05% trypsin, minimal incubation) to detach cells while preserving surface glycoproteins necessary for ConA binding.
Tissue Samples:
Process tissues to single-cell suspensions through mechanical dissociation or enzymatic digestion. Optimal methods vary by tissue type.
Immune Cells:
Use isolated nuclei or cross-linking strategies to avoid inadvertent immune activation by ConA beads.
Cross-linked Material:
Light cross-linking (0.1-1% formaldehyde, 1 minute) may improve signal for labile targets like histone acetylation, though it reduces overall yields.
Spike-in Controls for Normalization
E. coli Spike-in DNA:
Added during the stop buffer step to enable experimental normalization. Optimize amount based on cell input and target abundance.
SNAP-CUTANA K-MetStat Panel:
Semi-synthetic nucleosome spike-ins for histone methylation studies. These provide internal controls for antibody specificity, assay success, and quantitative normalization.
Frequently Asked Questions
What is the best way to assess CUT&RUN success before sequencing?
The most reliable indicators are: higher DNA yields in positive controls versus IgG negative controls, successful library preparation with expected yields (~300-500 ng from 5 ng input), and appropriate fragment size distribution (~300 bp peak) in library quality control traces. Avoid pre-library fragment analysis as it's not informative for CUT&RUN.
How does CUT&RUN compare to ChIP-seq for transcription factor mapping?
CUT&RUN provides higher resolution mapping with lower background and fewer cells. While some transcription factors generate sub-nucleosomal fragments (<120 bp), the protocol effectively captures these fragments. Library preparation modifications can further enhance representation of small fragments.
Can I use ChIP-validated antibodies for CUT&RUN?
Not necessarily. Antibody performance varies between techniques due to different accessibility and binding conditions. Some ChIP-validated antibodies work well, but others may not. Always include appropriate controls and consider using antibodies specifically validated for CUT&RUN.
What types of samples are compatible with CUT&RUN?
The protocol works with various samples including suspension cells, adherent cells, isolated nuclei, cryopreserved material, and lightly cross-linked samples. Tissue samples require processing to single-cell suspensions. Immune cells may need special handling to prevent activation.
How do I normalize CUT&RUN data between experiments?
Use spike-in controls like E. coli DNA or SNAP-CUTANA nucleosomes. For E. coli spike-ins, align reads to both the experimental genome and E. coli reference, then calculate normalization factors based on spike-in read percentages.
What sequencing depth is required for CUT&RUN experiments?
Typically only 3-8 million paired-end reads per sample are needed—significantly less than ChIP-seq. Low-abundance targets like H3K4me3 require 3-5 million reads, while high-abundance targets like H3K27me3 need 5-8 million reads.
Best Practices for Success
- Start with controls: Always include positive and negative controls, especially when working with new cell types or targets
- Optimize permeabilization: Perform digitonin titrations for each new cell type to achieve optimal permeabilization without lysis
- Handle beads carefully: Use nutators instead of rotators to prevent bead drying and sample loss
- Proceed directly to library prep: Avoid pre-library fragment analysis as yields are typically below detection limits
- Use appropriate spike-ins: Incorporate normalization controls for more reliable quantitative comparisons
- Validate antibodies: Use antibodies specifically validated for CUT&RUN or thoroughly test ChIP-validated antibodies
- Monitor sample quality: Implement quality control checks for cell integrity and bead binding throughout the protocol
CUT&RUN represents a significant advancement in chromatin profiling technology, offering higher resolution, lower background, and reduced input requirements compared to traditional methods. By following this optimized protocol and implementing appropriate controls, researchers can obtain high-quality genome-wide maps of chromatin features across various biological systems. 👉 Access additional protocol resources