---
title: "Cross-linking cells for CUT&Tag"
slug: "cross-linking-cut-and-tag"
updated: 2025-11-13T14:32:28Z
published: 2025-11-13T14:32:28Z
canonical: "support.epicypher.com/cross-linking-cut-and-tag"
---

> ## Documentation Index
> Fetch the complete documentation index at: https://support.epicypher.com/llms.txt
> Use this file to discover all available pages before exploring further.

# 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](https://www.epicypher.com/products/epigenetics-reagents-and-assays/cutana-chic-cut-and-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](https://support.epicypher.com/docs/trypan-blue-staining-cut-and-tag). Transfer 100,000 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](https://support.epicypher.com/docs/nuclei-extraction-cut-and-tag).
  2. For adherent cells: See [this article](https://support.epicypher.com/docs/adherent-cells-cut-and-tag) for instructions on collecting cells. Proceed to [nuclei extraction](https://support.epicypher.com/docs/nuclei-extraction-cut-and-tag).
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.

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