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.
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 for CUT&RUN (Day 1)
During Buffer Prep, supplement the Pre-Wash Buffer with 1% Triton X-100 and 0.05% SDS.
For CUTANA CUT&RUN Kit users, use the modified Pre-Wash Buffer to prepare Wash, Cell Permeabilization, and Antibody Buffer.
For DIY CUT&RUN Protocol users, use the modified Pre-Wash Buffer to prepare Wash, Digitonin, and Antibody Buffers.
Perform cross-linking prior to cell harvest, at the beginning of the Binding Cells to Activated Beads protocol section, 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) to a fresh tube.
For adherent cells, cross-linking will be performed while cells are still attached to the plate. Continue to next step.
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: 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.
For adherent cells: See this article for instructions on collecting cells.
Proceed with Day 1 as normal, using the modified Wash, Cell Permeabilization (Digitonin) and Antibody Buffers.
Cross-linking Protocol for CUT&RUN (Day 2)
Perform CUT&RUN Day 2 as normal, using the modified Wash, Cell Permeabilization (Digitonin) and Antibody Buffers.
At the end of the CUTANA CUT&RUN Kit or DIY protocol, collect CUT&RUN-enriched chromatin in new 8-strip tubes.
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. Purify CUT&RUN DNA following the CUTANA CUT&RUN Kit Protocol 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.