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 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.

Cross-linking Protocol for CUT&RUN (Day 1)

1. During Buffer Prep, supplement the Pre-Wash Buffer with 1% Triton X-100 and 0.05% SDS.

   1. For CUTANA CUT&RUN Kit users, use the modified Pre-Wash Buffer to prepare Wash, Cell Permeabilization, and Antibody Buffer.

   2. For DIY CUT&RUN Protocol users, use the modified Pre-Wash Buffer to prepare Wash, Digitonin, and Antibody Buffers.

2. Perform cross-linking prior to cell harvest, at the beginning of the Binding Cells to Activated Beads protocol section, 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) to a fresh tube.  

   2. For adherent cells, cross-linking will be performed while cells are still attached to the plate. Continue to next step.

3. 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.

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

5. Incubate for 1-10 min at room temperature (RT). 1 min is recommended. Test a range of times to determine optimal fixation conditions.

6. 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: Spin at 600 x g for 3 min at RT. Resuspend cells in CUT&RUN Wash Buffer and continue with sample prep, as directed in Binding Cells to Activated Beads in the Kit Manual or DIY Protocol.

   2. For adherent cells: See this article for instructions on collecting cells.

7. Proceed to binding cross-linked samples to ConA beads. Note that you will continue using the modified Wash, Cell Permeabilization (Digitonin) and Antibody Buffers.

Cross-linking Protocol for CUT&RUN (Day 2)

1. Perform CUT&RUN Section 5: Binding of pAG-MNase and Section 6: Targeted Chromatin Digestion and Release as normal. Use the modified Wash, Cell Permeabilization (Digitonin) and Antibody Buffers.

2. At the end of Section 6, CUT&RUN-enriched supernatants are in fresh 8-strip tubes. To reverse cross-links:

   1. Add 0.8 μL 10% SDS to each supernatant.

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

   3. Vortex to mix and quick spin to collect liquid.

   4. Place 8-strip tubes in a thermocycler set to 55˚C. Incubate overnight.

3. The next day, quick spin tubes. Purify CUT&RUN DNA following the Section 7 of the CUT&RUN Kit Manual or your preferred method. Quantify DNA and proceed to library prep.

Alternative cross-linking strategies

If the recommended CUT&RUN cross-linking protocol causes your cells to lyse and/or your reactions to become very sticky and clumpy, we suggest the following:

• Do NOT supplement the Pre-Wash Buffer with 1% Triton X-100 and 0.05% SDS. Use normal CUT&RUN assay buffers on Days 1 and 2.

• Do NOT reverse cross-links on Day 2. Just purify CUT&RUN-enriched DNA as per the standard protocol.

• Always run native materials in parallel.