CUT&RUN (Cleavage Under Targets and Release Using Nuclease) is an antibody-guided chromatin profiling method for mapping histone modifications, transcription factors and other DNA-associated proteins at high resolution. Instead of cross-linking chromatin, fragmenting all DNA and then immunoprecipitating material as in conventional ChIP, CUT&RUN recruits a Protein A/G–micrococcal nuclease fusion to an antibody-bound chromatin target inside permeabilised cells or nuclei. Calcium activation triggers local cleavage around the antibody-bound target, and the released fragments are recovered from the supernatant for qPCR or sequencing.
The method was introduced by Skene and Henikoff as a low-background, high-resolution alternative for chromatin profiling and is particularly attractive when sample input is limited. Because nuclease cleavage is physically tethered to the target, background from bulk chromatin can be much lower than in standard ChIP.
Important: CUT&RUN performance depends strongly on antibody compatibility, permeabilisation, nuclease activation, digestion time and sample type. The values below are a method-development framework, not universal reagent instructions.
Protocol at a glance
| Stage | Purpose | Critical variable |
|---|---|---|
| Cell/nuclei preparation | Preserve native chromatin while enabling reagent access | Sample integrity and cell number |
| Antibody binding | Target the chromatin feature of interest | Antibody specificity and native epitope access |
| pA/G–MNase recruitment | Position nuclease near antibody-bound chromatin | Fusion-protein binding and washing |
| Calcium activation | Cleave DNA adjacent to target | Temperature and digestion time |
| Fragment release | Separate target-associated fragments from bulk chromatin | Complete stopping and gentle handling |
| Library/QC | Convert released DNA into sequenceable library | Low-input DNA handling and PCR cycle count |
When to use CUT&RUN
- Map histone modifications with low background.
- Profile transcription-factor occupancy where a compatible antibody is available.
- Work with limited cell numbers compared with many conventional ChIP workflows.
- Compare chromatin occupancy between treatments, genotypes or developmental states.
- Generate high-resolution paired-end sequencing libraries.
- Validate selected loci by CUT&RUN-qPCR before committing to sequencing.
Equipment required
- Refrigerated centrifuge.
- Magnetic rack.
- Low-binding tubes.
- Tube rotator.
- Thermomixer or precisely controlled temperature block where required.
- Pipettes with low-retention tips.
- DNA purification system compatible with low-input fragments.
- Fragment analyser, Bioanalyzer or equivalent library-QC system for sequencing workflows.
Core reagents
- Fresh cells or isolated nuclei.
- Concanavalin A-coated magnetic beads.
- Permeabilisation buffer containing a mild detergent such as digitonin at an empirically optimised concentration.
- Target-specific primary antibody.
- Species-appropriate secondary antibody where needed to improve Protein A/G recruitment.
- Protein A/G–MNase fusion reagent.
- Calcium-containing activation solution.
- Stop buffer with chelator and stabilising components appropriate to the protocol.
- Protease inhibitors.
- DNA purification reagents.
- Indexed library-preparation reagents for sequencing.
Antibody selection
Antibody quality is one of the strongest determinants of CUT&RUN signal. Use an antibody with evidence for native chromatin recognition. ChIP or CUT&Tag validation can be useful background evidence, but neither automatically proves CUT&RUN compatibility. Cross-link-dependent epitopes may perform poorly in native CUT&RUN, and some antibodies that work in western blot recognise only denatured protein.
- Prefer antibodies supported by genetic or orthogonal specificity evidence.
- For histone marks, include a well-characterised positive-control mark during assay development.
- For transcription factors, confirm the protein is expressed in the chosen sample and include a known positive locus if available.
- Keep antibody lot consistent during comparative studies and record the lot number.
Detailed CUT&RUN protocol
- Prepare intact cells or nuclei. Harvest gently and remove culture medium. Avoid harsh handling that disrupts nuclei or releases genomic DNA.
- Bind to ConA magnetic beads. Equilibrate beads in the appropriate binding buffer, combine with cells/nuclei, and mix gently so samples are immobilised without excessive aggregation.
- Permeabilise. Transfer bead-bound samples into chilled permeabilisation/wash buffer. Digitonin concentration should be sufficient to permit antibody and fusion-protein entry while maintaining nuclear integrity.
- Add primary antibody. Incubate with the target-specific antibody at the validated concentration. Longer cold incubations can improve occupancy for some antibodies, but excessive incubation can increase background.
- Wash away unbound antibody. Use gentle magnetic separation and avoid losing bead-bound material.
- Add secondary antibody if required. This can improve pA/G recruitment for primary-antibody isotypes with weak Protein A/G binding. Keep the control design matched.
- Recruit pA/G–MNase. Incubate the antibody-bound chromatin with the fusion protein under non-activating conditions.
- Wash thoroughly. Remove unbound nuclease fusion. Residual free nuclease can increase non-specific cleavage.
- Chill the sample. Bring reactions to the validated low temperature before calcium activation. Temperature control helps restrict cleavage kinetics.
- Activate MNase with calcium. Add activation solution and incubate for the empirically determined interval. Use the same timing across samples.
- Stop the reaction. Add chelating stop buffer to terminate nuclease activity promptly.
- Release target-associated fragments. Incubate according to the selected protocol so cleaved chromatin fragments diffuse into the supernatant.
- Magnetically separate. Recover the DNA-containing supernatant without disturbing the bead/cell pellet.
- Purify DNA. Use a method that retains short fragments and minimises low-input loss.
- QC or qPCR. For assay development, compare positive locus, negative locus and IgG/no-antibody control before sequencing.
- Prepare indexed sequencing libraries. Minimise PCR amplification to what is needed for robust library recovery.
- Sequence paired-end. Paired-end reads help infer fragment length and improve high-resolution mapping.
Essential controls
- IgG or no-antibody control: estimates non-specific cleavage and fusion-protein background.
- Positive-control chromatin target: a robust histone modification or other established target can confirm that the workflow itself worked.
- Negative genomic locus: useful for qPCR and targeted QC.
- Biological negative control: knockout, depletion or a condition lacking target occupancy is particularly valuable for transcription-factor experiments.
- Optional spike-in: exogenous chromatin or DNA may support between-sample normalisation in selected experimental designs, but should be planned before the experiment rather than added as a reflex.
Optimising MNase digestion
Under-digestion reduces recovery and can leave target fragments trapped in the pellet. Over-digestion increases background, can erode resolution and may change the fragment-size distribution. Optimise activation time using a positive-control antibody and matched negative control before scaling to a sequencing experiment.
- Keep calcium addition and stopping time tightly synchronised.
- Use identical sample temperature during activation.
- Do not extrapolate nuclease time directly between different cell types or nuclei preparations.
- If background is high, first confirm wash quality and free-nuclease removal before shortening cleavage dramatically.
CUT&RUN-qPCR
Before sequencing a new target, CUT&RUN-qPCR can confirm locus-specific enrichment. Design primers around a known positive binding site and a genomic region expected to be negative. Report enrichment relative to input-like reference or control strategy chosen for the experiment, and include IgG/no-antibody background.
Sequencing and bioinformatic interpretation
- Inspect library concentration and fragment-size distribution before sequencing.
- Use paired-end alignment and retain fragment-size information.
- Evaluate replicate concordance rather than relying on one library.
- Compare target signal with matched IgG/no-antibody background.
- For narrow transcription-factor peaks, use analysis parameters suited to focal binding; broad histone marks require different peak models.
- Document genome build, aligner, duplicate handling and peak-calling parameters.
CUT&RUN versus ChIP-seq
| Feature | CUT&RUN | ChIP-seq |
|---|---|---|
| Chromatin treatment | Usually native or minimally fixed | Commonly formaldehyde cross-linked |
| Fragmentation | Targeted MNase cleavage | Bulk sonication or enzymatic fragmentation |
| Background | Often low | Can be higher from bulk chromatin |
| Input requirement | Can be low | Often higher |
| Resolution | High around targeted chromatin | Depends on fragment size and cross-linking |
| Antibody dependence | Very high | Very high |
Warnings and common analytical traps
- Do not equate ChIP validation with guaranteed CUT&RUN performance.
- Do not over-digest simply to increase DNA yield. More DNA can mean more background.
- Do not compare samples activated for different times or at different temperatures.
- Do not ignore bead loss. Losing ConA-bound cells produces apparent low signal unrelated to biology.
- Do not judge a low-input library by total DNA yield alone. Specific enrichment matters more than bulk DNA.
Limitations
- Requires a native-epitope-compatible antibody.
- Fragile or highly unusual nuclei can require customised permeabilisation.
- Very transient or fixation-dependent interactions may be difficult to preserve.
- Low-input sequencing libraries are vulnerable to amplification bias if over-cycled.
- Between-sample normalisation requires careful experimental design.
Troubleshooting
| Problem | Likely cause | What to try |
|---|---|---|
| Low recovery | Poor antibody binding, inadequate permeabilisation or under-digestion | Confirm target expression, optimise detergent and antibody, and run a timed MNase optimisation. |
| High IgG background | Free nuclease, poor washing or damaged nuclei | Improve washes, reduce sample damage and review fusion-protein concentration. |
| Broad/non-specific signal | Over-digestion or non-specific antibody | Shorten activation and validate antibody specificity with biological controls. |
| Library dominated by very short fragments | Over-cleavage or library artefact | Reduce digestion and review size-selection/amplification conditions. |
| Poor replicate agreement | Variable bead capture, timing or cell state | Standardise cell input, ConA binding, calcium activation and biological preparation. |
Frequently asked questions
How many cells are needed for CUT&RUN?
Published CUT&RUN workflows can operate with substantially fewer cells than many conventional ChIP protocols, but the minimum depends on target abundance, antibody performance and library method. Optimise with the actual target rather than assuming a universal minimum.
Does CUT&RUN require cross-linking?
No. One advantage is that it can profile native chromatin. Selected targets or sample types may benefit from mild fixation, but fixation changes accessibility and must be validated.
Can I use a ChIP antibody?
It is a reasonable candidate if the epitope remains accessible in native chromatin, but validate it directly in CUT&RUN.
References and journal sources
- Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife. 2017;6:e21856. doi:10.7554/eLife.21856.
- Meers MP et al. High-Resolution Chromatin Profiling Using CUT&RUN. Curr Protoc Mol Biol. 2019. PMID:30688406.
Relevant ABMIUM products
Antibody application evidence must be matched to CUT&RUN specifically. ABMIUM's Histone H3 Antibody [B552] is listed for ChIP and CUT&Tag, so it is a related chromatin-profiling example, not an automatically validated CUT&RUN antibody. For another target, use Find your MATCH and we can shortlist antibodies with the strongest native chromatin evidence.
Explore more with ABMIUM
Compare this workflow with the ChIP Protocol and the CUT&Tag Protocol, or explore chromatin-related antibodies.