---
title: "Nuclei extraction protocol for CUTANA™ assays"
slug: "nuclei-extraction-cut-and-tag"
updated: 2026-06-16T19:35:33Z
published: 2026-06-16T19:35:33Z
canonical: "support.epicypher.com/nuclei-extraction-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.

# Nuclei extraction protocol for CUTANA™ assays

> [!NOTE]
> This article also applies to meCUT&RUN and Multiomic CUT&RUN.
> 
> Important Notes
> 
> - Nuclei Extraction Buffer is prepared fresh on the day of nuclei harvest.
> - The CUTANA™ Nuclei Extraction Protocol is optimized for CUT&RUN and CUT&Tag workflows. For CUT&RUN, start with 500,000 cells per reaction; for CUT&Tag, start with 100,000 cells per reaction. Harvest 10% excess cells to account for sample loss.
> - Reconstitute Protease Inhibitor Tablets with molecular biology-grade water to make a 25X stock. If using [EpiCypher Protease Inhibitor Tablets](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-protease-inhibitor-tablets), follow reconstitution instructions on product page or technical data sheet.
> - The Pre-Nuclei Extraction Buffer and prepared Nuclei Extraction Buffer are very viscous; pay careful attention to preparation notes for both to ensure that they are adequately mixed.

## **Materials Needed**

| Reagent | Source |
| --- | --- |
| Pre-Nuclei Extraction Buffer | [EpiCypher 21-1026](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-nuclei-extraction-buffer) |
| 1M Spermidine |
| Protease Inhibitor Tablets, EDTA-free | [EpiCypher 21-1027-S or 21-1027-L](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-protease-inhibitor-tablets) |
| Phosphate Buffered Saline (PBS) | Any vendor |
| 0.4% Trypan Blue | Invitrogen T10282 |
| Brightfield or phase microscope + hemacytometer slides | Any vendor |

## **Nuclei Prep Protocol**

1. Prepare Nuclei Extraction Buffer in a fresh tube. Per reaction, combine 235 µL Pre-Nuclei Extraction Buffer, 0.13 µL Spermidine, and 9.8 µL 25X Protease Inhibitor.
  - If preparing buffer for fewer than 8 reactions, dilute the 1M Spermidine stock 1:10 in molecular biology-grade water and add 1.3 μL per reaction.
  - **Note:** The Pre-Nuclei Extraction Buffer is very viscous. Mix well prior to combining (ideally using a pipette, as vortexing can cause bubbles).
2. Place final Nuclei Extraction Buffer on ice.
3. Count fresh or frozen cells and confirm starting cell integrity, morphology, and viability by [Trypan Blue staining](/v1/docs/trypan-blue-staining). It is important that cells have good starting viability. For K562 cells, we aim for >90% viability.
4. Harvest cells. For CUT&RUN, harvest 500,000 cells per reaction; for CUT&Tag, harvest 100,000 cells per reaction. Harvest 10% excess cells to account for sample loss.
5. Spin cells at 600 x g for 3 min at room temperature (RT). Remove supernatant and resuspend cells in 100 µL per reaction cold Nuclei Extraction Buffer. **Pipette mix the Nuclei Extraction Buffer thoroughly before resuspending.**
6. Incubate on ice for 10 min.
7. Spin at 600 x g for 5 min at 4ºC. Remove and discard supernatant. The pellet should change in appearance from sticky, pale yellow (cells) to white and fluffy (nuclei).
8. PLEASE NOTE: This step varies depending on which assay you are performing:
  - In CUT&RUN: Gently resuspend nuclei in 105 µL per reaction cold **Wash Buffer.**
  - In CUT&Tag: Gently resuspend nuclei in 105 µL per reaction cold **Wash Buffer 1.**
  - If cryopreserving (for either assay): Gently resuspend nuclei in 105 uL per reaction cold **Nuclei Extraction Buffer** and skip to **Nuclei Preservation and Thawing Protocol** below.
9. Take a 10 µL aliquot to examine nuclear integrity by [Trypan Blue staining](/v1/docs/trypan-blue-staining). Isolated nuclei will stain Trypan Blue positive and should have clear borders and minimal debris. Intact cells will be bright white and round (see **Figure** **1**). For accurate nuclei counts, record "dead" cell numbers on an automated cell counter or manually count blue stained nuclei.
10. Proceed to desired assay ([CUT&RUN](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-chic-cut-run-kit) or [CUT&Tag](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-cut-and-tag-kit))—**nuclei in Wash Buffer/Wash Buffer 1 can be directly added to activated ConA beads.** If freezing nuclei for later use, see cryopreservation protocol below.

![nuclei extraction NEW](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/nuclei%20extraction%20NEW.png)

**Figure 1.** Nuclei successfully isolated with CUTANA Nuclei Extraction Protocol. Viable starting cells exclude Trypan Blue (top row), while isolated nuclei stain Trypan Blue positive (bottom row).

## **Nuclei Cryopreservation and Thawing Protocol**

1. Aliquot nuclei resuspended in Nuclei Extraction Buffer. EpiCypher scientists typically aliquot for >8 reactions (800 µL + plus 20-30% excess) to account for sample loss at thaw steps.
2. Slowly freeze aliquots in an isopropanol-filled chiller in a -80°C freezer.
3. When ready to use samples, thaw nuclei by placing on ice for 5-10 minutes. Move quickly to avoid nuclear lysis and chromatin fragmentation. Note that only nuclei which have been in the -80°C freezer for at least 24 hours are recommended for thawing.
4. Thawed nuclei in Nuclei Extraction Buffer can be directly added to activated ConA beads in both [CUT&RUN](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-chic-cut-run-kit) and [CUT&Tag](https://www.epicypher.com/products/epigenetics-kits-and-reagents/cutana-cut-and-tag-kit).