---
title: "Cross-Linking for CUT&RUN | Protocol & Best Practices"
slug: "cross-linking-for-cut-and-run"
description: "Learn when to use cross-linking for CUT&RUN, recommended fixation conditions, protocol tips, and how to preserve assay performance."
tags: ["cut and run"]
updated: 2026-06-11T15:48:46Z
published: 2026-06-11T15:48:46Z
canonical: "support.epicypher.com/cross-linking-for-cut-and-run"
---

> ## 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&RUN

> [!NOTE]
> This article also applies to meCUT&RUN and Multiomic 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 preserves signal without negatively impacting data.
- If light cross-linking is not sufficient, moderate cross-linking (1% formaldehyde, 1 min) can be used with the caveat that it may reduce DNA yield.
- Avoid heavy cross-linking conditions used for ChIP (>1% formaldehyde, 1-10 min), which negatively impacts CUT&RUN DNA yield and data quality.

## **Cross-linking Protocol for CUT&RUN**

| Materials Needed | Source |
| --- | --- |
| 37% Formaldehyde | EMS 15686 |
| Glycine | Sigma 50046 |
| 20 μg/μL Proteinase K | Ambion AM2546 |

1. Collect cells for CUT&RUN:
  1. Suspension culture cells: Make sure cells are well mixed and take a 10 µL aliquot to count using [Trypan Blue staining](/v1/docs/trypan-blue-staining). Transfer 500,000 cells per reaction (plus 10% excess cells) to a fresh tube.
  2. Adherent cells: cross-linking will be performed while cells are still attached to the plate. Continue to next step.
2. Cross-link cells:
  - 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 min at room temperature (RT).
3. Quench cross-linking:
  - Add 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.
  - Adherent cells: See [this article](/v1/docs/sample-prep-for-adherent-cells) for instructions on collecting cells.
4. Proceed to washing cells in CUT&RUN buffers and binding to ConA beads.

**No other modifications are required for the CUT&RUN protocol**. You do not need to reverse cross-links.

## **Alternative Cross-linking Strategy**

If the standard cross-linking protocol does not work for your sample, we recommend trying this alternative strategy for cross-linking samples in CUT&RUN.

1. CUT&RUN Buffer Preparation: Supplement the Pre-Wash Buffer with 1% Triton X-100 and 0.05% SDS.
  - For CUTANA CUT&RUN Kit users: Use this modified buffer to prepare Wash, Cell Permeabilization, and Antibody Buffers.
  - For DIY CUT&RUN Protocol users: Use this modified buffer to prepare Wash, Digitonin, and Antibody Buffers.
2. Cross-linking: Collect cells and follow the cross-linking workflow outlined in **Day 1** above.
3. Day 2 Adjustments: After Section 6 (Targeted Chromatin Digestion and Release), reverse cross-links before DNA purification:
  - Add 0.8 μL 10% SDS to each supernatant.
  - Add 1 μL 20 μg/μL Proteinase K to each supernatant.
  - Vortex to mix and quick spin to collect liquid.
  - Incubate overnight at 55°C.
  - The next day, quick spin tubes to collect liquid. Purify CUT&RUN DNA.