Cross-linking cells for CUT&Tag

Important Notes

In this article, you will find guidance for incorporating cross-linking into your CUT&Tag experiment. We recommend cross-linking prior to nuclei extraction in order to best preserve in vivo interactions. ALWAYS include native samples when testing cross-linking conditions.

While cross-linking is not necessary for CUT&Tag, it may be beneficial for labile targets (such as histone lysine acetylation) or experiment with tightly controlled time points.

Note that for transiently interacting chromatin-associated proteins, it is recommended to use the CUTANA™ CUT&RUN Kit, which includes methods for light to moderately cross-linked samples.

How to optimize cross-linking for CUT&Tag

  • 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

37% Formaldehyde

EMS 15686

Glycine

Sigma 50046

Cross-linking Protocol for CUT&Tag

  1. Perform cross-linking prior to cell harvest, at the beginning of the Nuclei Prep and Binding to Beads protocol section.

    1. For suspension culture cells, make sure cells are well mixed. 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.

  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 plate (adherent cells) to mix.

    1. Suspension cells: Spin at 600 x g for 3 min at RT. Remove supernatant and proceed to nuclei extraction.

    2. For adherent cells: See this article for instructions on collecting cells. Proceed to nuclei extraction.

  6. No additional protocol modifications are required. Digestion with SDS Release Buffer on Day 2 of the CUT&Tag protocol is sufficient to reverse cross-links.

  7. Select the optimal sample prep conditions for the target based on the balance of DNA yield, enrichment, and signal-to-noise in sequencing data.