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 |
CROSS-LINKING PROTOCOL
CUT&RUN DAY 1
- Prepare CUT&RUN Wash, Cell Permeabilization, and Antibody Buffers using Pre-Wash Buffer supplemented with 1% Triton X-100 and 0.05% SDS.
- Perform cross-linking at the beginning of Protocol: Section III as follows:
- 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.
- For adherent cells, cross-linking will be performed while cells are still attached to the plate.
- 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. Test a range of times to determine optimal fixation conditions.
- Quench cross-linking by adding Glycine to a final concentration of 125 mM. Vortex (suspension cells) or swirl (adherent cells) to mix.
- Suspension cells: Proceed to Protocol: Section III Step 2 of the CUT&RUN protocol (spin at 600 x g for 3 min at RT).
- For adherent cells: See this article for instructions.
CUT&RUN DAY 2
- Following the collection of supernatants containing CUT&RUN-enriched DNA (Protocol: Section VI), it is crucial to reverse cross-links.
- 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.
- Place supernatants (in 8-strip tubes) in a thermocycler set to 55˚C. Incubate overnight.
- The next day, quick spin tubes and resume CUT&RUN at Section VII: DNA Purification (columns or beads).
