CUT&RUN Protocol v2.0: Genomic Profiling of Histone PTMs and Chromatin-Associated Proteins

Overview

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a high-resolution genomic mapping technique for profiling histone post-translational modifications (PTMs), transcription factors (TFs), and chromatin regulators. Developed by Dr. Steven Henikoff, this protocol leverages pAG-MNase to cleave antibody-bound chromatin in situ, offering advantages over traditional ChIP-seq, including lower background, reduced cell input, and higher signal-to-noise ratios.

Key Workflow Steps

1. ConA Bead Activation: Immobilize cells/nuclei to Concanavalin A-coated magnetic beads.
2. Antibody Binding: Incubate with target-specific antibodies (overnight at 4°C).
3. pAG-MNase Recruitment: Bind fusion protein to antibody-labeled chromatin.
4. Chromatin Digestion: Activate MNase with Ca²⁺ to release target DNA fragments.
5. Library Preparation: Purify DNA and prepare Illumina sequencing libraries.

Advantages of CUTANA™ CUT&RUN

• Low Input: Requires only 5,000–500,000 cells per reaction.
• High Sensitivity: Detects low-abundance targets (e.g., H3K4me3) with 3–8 million sequencing reads.
• Versatility: Compatible with native, fixed, or frozen cells/nuclei.
• Spike-in Controls: Includes SNAP-CUTANA™ K-MetStat Panel for normalization and antibody validation.

Experimental Design

Essential Controls

• Negative Control: Rabbit IgG (EpiCypher 13-0042).

• Positive Controls:

  • H3K4me3 (low-abundance PTM).
  • CTCF or BRD4 (chromatin-associated proteins).
• Spike-ins: Add E. coli DNA (1% of total reads) or SNAP-CUTANA™ nucleosomes for quantitative normalization.

Optimization Tips

• Digitonin Permeabilization: Titrate (0.001–0.1%) to achieve >95% cell permeability.
• Cell Input: Start with 500,000 K562 cells; scale down to 50,000 cells for validated targets.

Protocol Highlights

Section I: ConA Bead Activation

1. Resuspend ConA beads in Bead Activation Buffer (20 mM HEPES, 10 mM KCl, 1 mM CaCl₂/MnCl₂).
2. Wash twice, then resuspend in ice-cold buffer for cell binding.

Section II: Cell Binding

1. Harvest cells, wash in PBS, and bind to activated ConA beads (10 min at RT).
2. QC Check: Confirm >90% cell viability via Trypan blue exclusion (Figure 6).

Section V: Chromatin Digestion

1. Quench MNase with Stop Buffer (340 mM NaCl, 20 mM EDTA, RNase A).
2. Purify DNA using CUTANA™ DNA Purification Kit (retains fragments >50 bp).

FAQs

1. How to assess success before sequencing?

• QC Metrics:

  • DNA yield: Positive control > IgG.
  • TapeStation: Expect ~300 bp peaks (nucleosomes + adapters).
• Avoid pre-library fragment analysis (low DNA yields).

2. Can ChIP antibodies be used?

Some ChIP antibodies work, but CUTANA-validated antibodies (e.g., H3K4me3, CTCF) are recommended.

3. Compatible sample types?

• Adherent cells: Use mild trypsinization (0.05%, 37°C).
• Tissues: Mechanically dissociate or enzymatically digest.
• Immune cells: Isolate nuclei or cross-link to prevent ConA stimulation.

Library Preparation & Sequencing

• Kit: CUTANA™ Library Prep Kit (48-plex indexing).
• Sequencing: Illumina (3–8M paired-end reads/sample). Pool libraries equimolarly.
• Analysis: Align to reference genome + E. coli (for spike-in normalization).

👉 Explore CUTANA™ CUT&RUN reagents

References

1. Skene & Henikoff (2017). eLife 6:e21856.
2. EpiCypher SNAP-CUTANA™ Spike-in User Guide.

For protocol updates: epicypher.com/protocols

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