Cross-linking cells for CUT&RUN

Important Notes

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

  • Transient chromatin interactions (such as acetyl-lysine reader proteins or remodeling enzymes).

How to Optimize Cross-linking for CUT&RUN

  • Start with light cross-linking (0.1% formaldehyde, 1 min), which preserves signal without negatively impacting data.

  • If light cross-linking is not sufficient, moderate cross-linking (1% formaldehyde, 1 min) can be used with the caveat that it may reduce DNA yield.

  • Avoid heavy cross-linking conditions used for ChIP (>1% formaldehyde, 1-10 min), which negatively impacts CUT&RUN DNA yield and data quality.

Cross-linking Protocol for CUT&RUN

Materials Needed

Source

37% Formaldehyde

EMS 15686

Glycine

Sigma 50046

20 μg/μL Proteinase K

Ambion AM2546

  1. Collect cells for CUT&RUN:

    1. 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 cells) to a fresh tube.  

    2. Adherent cells: cross-linking will be performed while cells are still attached to the plate. Continue to next step.

  2. Cross-link cells:

    • 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.

    • Quickly vortex (suspension cells) or swirl plate (adherent cells) to mix.

    • Incubate for 1-10 min at room temperature (RT). 1 min is recommended.

  3. Quench cross-linking:

    • Add Glycine to a final concentration of 125 mM. Vortex (suspension cells) or swirl (adherent cells) to mix.

    • Suspension cells: Spin at 600 x g for 3 min at RT. Resuspend cells in CUT&RUN Wash Buffer and continue with sample prep.

    • Adherent cells: See this article for instructions on collecting cells.

  4. Proceed to washing cells in CUT&RUN buffers and binding to ConA beads.

No other modifications are required for the CUT&RUN protocol.

Alternative Cross-linking Strategy

If the standard cross-linking protocol does not work for your sample, we recommend trying this alternative strategy for cross-linking samples in CUT&RUN.

  1. CUT&RUN Buffer Preparation: Supplement the Pre-Wash Buffer with 1% Triton X-100 and 0.05% SDS.

    • For CUTANA CUT&RUN Kit users: Use this modified buffer to prepare Wash, Cell Permeabilization, and Antibody Buffers.

    • For DIY CUT&RUN Protocol users: Use this modified buffer to prepare Wash, Digitonin, and Antibody Buffers.

  2. Cross-linking: Collect cells and follow the cross-linking workflow outlined in Day 1 above.

  3. Day 2 Adjustments: After Section 6 (Targeted Chromatin Digestion and Release), reverse cross-links before DNA purification:

    • Add 0.8 μL 10% SDS to each supernatant.

    • Add 1 μL 20 μg/μL Proteinase K to each supernatant.

    • Vortex to mix and quick spin to collect liquid.

    • Incubate overnight at 55°C.

    • The next day, quick spin tubes to collect liquid. Purify CUT&RUN DNA.