Cross-linking for CUT&RUN

While cross-linking is not necessary for CUT&RUN, it may be beneficial for:

  • Labile targets (such as histone lysine acetylation).
  • Experiments with tightly controlled time points.
  • Transiently chromatin-interacting proteins (such as acetyl-lysine reader proteins or remodeling enzymes).

In this article, you will find guidance for incorporating cross-linking into your CUT&RUN experiment. Importantly, ALWAYS include native samples when testing cross-linking conditions.

When optimizing cross-linking conditions:

  • Start with light cross-linking (0.1% formaldehyde, 1 min), which generally preserves signal without negatively impacting data.
  • If light cross-linking is not sufficient, moderate cross-linking (1% formaldehyde, 1 min) can be attempted with the caveat that it may reduce DNA yield.
  • Avoid heavy cross-linking conditions used for ChIP (>1% formaldehyde, 1-10 min) which is deleterious to both DNA yield and data quality.
Materials needed Source
Pre-Wash Buffer EpiCypher CUTANA CUT&RUN Kit 14-1048
Triton X-100, 10% solution Any vendor
SDS, 10% solution Any vendor
37% Formaldehyde Sigma 252549
Glycine Sigma 50046
20 μg/μL Proteinase K Ambion AM2546



  • Prepare CUT&RUN Wash, Cell Permeabilization, and Antibody Buffers using Pre-Wash Buffer supplemented with 1% Triton X-100 and 0.05% SDS.
  1. Perform cross-linking at the beginning of Protocol: Section III as follows:
    1. For suspension culture cells, make sure cells are well mixed and take a 10 µL aliquot to count using Trypan Blue staining. Transfer 500,000 cells per reaction (plus 10% excess) into a 15 mL or 1.5 mL tube.  
    2. For adherent cells, cross-linking will be performed while cells are still attached to the plate. 
  2. Add fresh 37% Formaldehyde directly to culture for a final concentration of 0.1-1%. Test a range of concentrations to optimize for target and cell type.
  3. Quickly vortex (suspension cells) or swirl plate (adherent cells) to mix.
  4. Incubate for 1-10 min at room temperature (RT). 1 min is recommended. Test a range of times to determine optimal fixation conditions.
  5. Quench cross-linking by adding Glycine to a final concentration of 125 mM. Vortex (suspension cells) or swirl (adherent cells) to mix.
    1. Suspension cells: Proceed to Protocol: Section III Step 2 of the CUT&RUN protocol (spin at 600 x g for 3 min at RT).
    2. For adherent cells: See this article for instructions.


  • Following the collection of supernatants containing CUT&RUN-enriched DNA (Protocol: Section VI), it is crucial to reverse cross-links.
  1. Add 0.8 μL 10% SDS followed by 1 μL of 20 μg/μL Proteinase K to each supernatant. Vortex to mix and quick spin to collect liquid.
  2. Place supernatants (in 8-strip tubes) in a thermocycler set to 55˚C. Incubate overnight.
  3. The next day, quick spin tubes and resume CUT&RUN at Section VII: DNA Purification (columns or beads).

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