CUT&RUN Overview

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is an epigenomic mapping assay that uses a Protein A/G Micrococcal Nuclease fusion protein (pAG-MNase) to selectively cleave antibody-bound chromatin. The entire assay takes place in intact cells or nuclei bound to magnetic beads, a solid support that streamlines sample processing. Following MNase activation, clipped chromatin fragments diffuse into solution while cells are easily removed using magnetic beads – eliminating a major source of background signal. Target-enriched DNA is then purified from the solution and used to prepare sequencing libraries.

CUT&RUN allows high-resolution epigenomic mapping for histone PTMs and chromatin-associated proteins using a small number of cells and only 3-8 million sequencing reads per reaction. The streamlined workflow and cost savings make CUT&RUN amenable to greater experimental throughput, allowing deeper and more rapid investigations to uncover epigenetic biology

How does CUT&RUN work?

CUT&RUN requires a few basic steps:

The main difference between ChIP-seq and CUT&RUN is how chromatin fragments are enriched. ChIP-seq (Chromatin ImmunoPrecipitation and sequencing) uses antibodies to “pull-down” target-associated DNA from a large pool of fragmented chromatin, resulting in high background and variable yields. In contrast, CUT&RUN uses pAG-MNase to selectively cleave antibody-bound chromatin in intact cells. Target-bound DNA diffuses into solution, leaving background behind – resulting in an assay with improved signal-to-noise and sensitivity.

What are the challenges of ChIP-seq?

ChIP-seq is a time-intensive and expensive technique with multiple problematic steps:

• Crosslinking: required to stabilize targets on chromatin, but causes artifacts
• Cellular lysis: makes samples viscous, hard to work with, and increases background
• Chromatin fragmentation: laborious, requires optimization, introduces variability
• Immunoprecipitation: requires stringent washes, diminishing signal-to-noise

To overcome these challenges, ChIP-seq requires high numbers of starting cells, deep sequencing, and complicated optimization steps. These requirements and the associated costs have precluded chromatin mapping for many applications, such as clinical and translational research, and slowed the development of effective epigenetics-targeted therapeutics.

CUTANA™ CUT&RUN profiling using 0.5 million K562 cells and < 4 million sequencing reads generates superior genomic maps for markers of silenced genes and active promoters

All of these challenges are eliminated in CUT&RUN.

Compared to ChIP-seq, CUT&RUN delivers superior genomic mapping data using up to 40-fold fewer cells and >10-fold reduced sequencing depth (below). The user-friendly CUT&RUN workflow can go from cells to sequencing libraries in just 3 days and requires minimal optimization steps, all at a fraction of the costs of ChIP-seq.

CUT&RUN excels where ChIP-seq fails – providing a fast, reliable assay compatible with multiple sample and target types. Learn more about the capabilities of CUT&RUN below.

Interested in CUT&RUN but have questions about how it will integrate into your studies? Read on.

I am new to genomics, is CUT&RUN for me?

Yes! CUT&RUN is a robust, reliable, user-friendly genomic mapping assay. Our CUTANA CUT&RUN and Library Prep kits provide everything you need to integrate CUT&RUN in your research.

Does CUT&RUN require cross-linking?

No, unlike ChIP-seq, CUT&RUN does not require cross-linking. The preferred input for CUT&RUN is native (unfixed) samples. In fact, heavy cross-linking as with ChIP-seq severely reduces CUT&RUN yields and is not recommended for CUT&RUN under any circumstances.

However, EpiCypher has found that light cross-linking can improve experimental success for some labile histone PTMs, such as acetylation, or other chromatin-associated proteins. Full experimental guidance on how to test cross-linking for a specific target can be found on our protocols page.

What sample types is CUT&RUN compatible with?

CUTANA CUT&RUN is compatible with a vast array of cell types:

Species: Human, mouse, Drosophila, zebrafish, and more

Cell processing: Cultured cells (adherent and suspension), FACS-isolated cells, frozen samples

Cell types: Cell lines, differentiated pluripotent stem cells, primary cells (e.g. immune cells*), whole tissues (e.g. brain)

*Note on immune cells: ConA is a lectin and may activate some immune cell types. We recommend using nuclei if performing CUT&RUN with immune cells.

For examples, see Key Publications and Example Applications below. Guidance on sample prep, including protocol modifications, can be found in the Tech Support Center.

Is CUT&RUN compatible with whole tissues?

Yes, CUT&RUN is compatible with tissues. Tissues must first be processed into a single-cell suspension by mechanical or enzymatic means before being bound to ConA beads. The Tech Support Center provides more guidance on these methods.

How many cells do you need for CUT&RUN?

EpiCypher’s CUTANA CUT&RUN assays are validated for 500,000 to 5,000 cells*. Note that some targets that provide robust CUT&RUN data at 500,000 may require additional optimization or even fail at low inputs. Full considerations for low-input CUT&RUN experiments can be found on the Tech Support Center.

*Using fewer than 5,000 cells comes with many caveats, including low yields, increased adapter dimer contamination in libraries, and increased sequencing depth requirements.

Will CUT&RUN work for my ChIP target?

Yes, most likely. To date, we’ve confirmed that all major target classes – including histone PTMs, transcription factors, chromatin readers, writers, and modifying enzymes (erasers, remodelers) – generate robust data in CUT&RUN, all you need is a good antibody!

Can I use my ChIP-validated antibody for CUT&RUN?

No, we do not recommend using ChIP-validated antibodies for CUT&RUN as they may perform poorly in CUT&RUN due to the differences between these two assays. In general, EpiCypher has found that antibody validation in ChIP, immunoblot, ELISA or any other method does not predict performance in CUT&RUN. The only way to know if your antibody works for CUT&RUN is to test it in CUT&RUN.

EpiCypher offers a suite of CUT&RUN-validated antibodies for histone PTMs and chromatin associated proteins. If we do not offer a CUT&RUN-validated antibody for your target, see the advice in this blog post or contact us.

Should I use CUT&RUN or CUT&Tag?

EpiCypher has extensive experience in developing CUT&RUN, CUT&Tag, and ChIP-seq assays. CUT&RUN is our all-purpose chromatin mapping assay as it balances cell input and sequencing requirements with versatile target compatibility, including chromatin-associated proteins. For a deeper discussion of what to consider when choosing between CUT&RUN and CUT&Tag, see this blog post.

Foundational CUT&RUN papers

CUTANA CUT&RUN in action