markdown-output/a-versatile-nuclei-extraction-protocol-for-single-crw4v7gw.md

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# Goal/Experiment:
Develop a versatile nuclei extraction protocol for single-cell multimodal ATAC and gene expression analysis in non-model species to ensure high-quality nuclei isolation for subsequent molecular biology applications.

# A Versatile Nuclei Extraction Protocol for Single Cell Multimodal ATAC and Gene Expression in Non-Model Species

**Authors:**
Rose Ruiz Daniels<sup>1</sup>, Richard S Taylor<sup>1</sup>, Ioannis Konstantinidis<sup>2</sup>, Sarah Salisbury<sup>1</sup>, Diego Perojil Morata<sup>1</sup>, Jorge Manuel de Oliveira Fernandes<sup>2</sup>, Emily Clark<sup>1</sup>, Dan Macqueen<sup>1</sup>, Diego Robledo<sup>1</sup>
**Affiliations:**
1. The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh EH25 9RG, UK.
2. Nord University, Bodø, Hovedbygning 2060, Norway.

**DOI:** [10.17504/protocols.io.bp2l69wnrlqe/v1](https://doi.org/10.17504/protocols.io.bp2l69wnrlqe/v1)

## Abstract

This protocol presents a modified version of an approach to extract nuclei from various tissue types for single-cell sequencing and aims to extract high-quality nuclei for single-cell multimodal ATAC and gene expression analysis using the 10x Chromium system. Key modifications include varied RNase inhibitor concentrations, nuclear isolation buffer quantities, and specific steps for multimodal analysis. 

For bulk ATAC-seq, use protease inhibitor cocktail PIC instead of RNase inhibitor in the snRNA-seq version of this protocol.

### Goal
Develop a high-quality nuclei extraction protocol from diverse tissue types for single-cell multi-omic applications ensuring optimal nuclei integrity and yield.

## Guidelines

**Trial Preparation:**
Conduct a trial run before library preparation, especially with new tissue types, to adjust parameters (e.g., mincing times, filter size) and optimize nuclei quality. Adjusting these parameters helps prevent RNAse introduction and ensures the production of nuclei with intact membranes.

## Materials

### Equipment and Supplies
- **Noyes Spring Scissors - Tungsten Carbide** (Fine Science Tools Catalog #15514-12)
- **Tungsten Carbide Straight Scissors 11.5 cm** (Fine Science Tools Catalog #14558-11)
- **40 µm Falcon™ Cell Strainers** (Fisher Scientific Catalog #08-771-2)
- **Corning™ Falcon™ Test Tube with 35 µm Cell Strainer Snap Cap** (Corning Catalog #352235)
- **pluriStrainer Mini 20 µm (Cell Strainer)** (pluriSelect Catalog #43-10020-50)
- X500 Eppendorf DNA LoBind Tubes, 1.5ml, PCR clean
- Cryotube
- 6-well tissue culture plate (Stem Cell Technologies)
- Falcon tubes 15 ml (Corning)
- **INCYTO C-Chip™ Disposable Hemacytometers** (VWR International Catalog #82030-468)

## Sampling and Storage for Nuclear Isolation

### Sampling
- **Euthanize and Process:** Euthanize animals humanely and immediately process the tissues.
- **Sample Preparation:** Freeze ~60 mg of sammond tissue in a cryotube and flash freeze in liquid nitrogen. If liquid nitrogen is unavailable, use dry ice. The tissue preservation step is critical.
- **Storage:** Store tissue samples at -80°C for up to one year. Older samples may still yield viable nuclei but require testing.

### Critical Steps
- **Immediate Freezing:** Preserve tissue samples as quickly as possible to maintain nuclear integrity.

## Reagents

Chill all reagents on ice before use.

**2X Stock of Salt-Tris Solution (10 mL)**

| Component             | Catalog Number                             | Stock Solution | Volume | Final Concentration |
|-----------------------|--------------------------------------------|----------------|--------|---------------------|
| NaCl (5 M) RNase-free | Thermo Fisher Scientific Catalog #AM9759   | 292 µL         |        | 146 mM              |
| Tris-HCl pH 7.5       | Thermo Fisher Catalog #15567027            | 100 µL         |        | 10 mM               |
| CaCl2 1 M Solution    | VWR International Catalog #97062-820       | 10 µL          |        | 1 mM                |
| MgCl2 1.00 M Solution | MilliporeSigma (Sigma-Aldrich) Catalog M1028 | 210 µL         |        | 21 mM               |
| Nuclease-free Water   | VWR International Catalog #97062-794       | 9388 µL        |        |                     |

### RNase Inhibitor-Containing Buffers

**Protector RNase Inhibitor** (Merck MilliporeSigma Catalog #3335399001)

**1X ST Buffer Solution**
- Dilute 2X ST with equal volumes of nuclease-free water (1:1) to create the 1X solution.
- 250 µL RNase inhibitor (200 U/mL) per 1X ST solution (10 mL).

**Working Solutions:**
1X ST with additional components:
  
| Component              | Catalog Number                  |  Volume | Final Concentration |
|------------------------|---------------------------------|---------|---------------------|
| 2X ST Buffer           |                                 |   2 mL  |                     |
| 1% Tween-20            | Sigma-Aldrich Catalog #P-7949   | 120 µL  |                     |
| 2% BSA                 | NEB Biolabs Catalog #B9000S     |  20 µL  |                     |
| Nuclease-free water    |                                 | 1810 µL |                     |
| RNase inhibitor        |                                 |  50 µL  | 1000 U/mL           |

Prepare and chill solutions immediately before use.

## Nucleus Isolation Workflow for ST-based Buffers

### Step 1: Initial Sample Preparation (30m)
- Keep samples frozen on dry ice until nuclei isolation.
- Pre-chill the centrifuge to 4°C.
- Place frozen tissue into a 6-well plate with 1 mL TST buffer.
- If tissue sticks to the cryotube, transfer using tweezers on dry ice.

### Step 2: Mincing (10m)
- Mince tissue with Tungsten Carbide scissors for 30 seconds, then switch to Noyes Spring Scissors for total of 10 minutes for hard tissues.
- For soft tissues, use spring scissors only for 10 minutes.

#### Step 2.1: Dissociation
- Pipette tissue gently using a P1000 pipette with a low-retention tip.
- Critical Step: Assess timing by examining nuclei under a microscope.
    - **Head Kidney Nuclei:** Insufficient dissociation time - not ideal.
    - **Blood Nuclei:** Perfect dissociation - ideal.
    - **Liver Nuclei:** Over-dissociation - not ideal.

### Step 3: Lysate Processing
- Pass lysate through a 40 µm cell strainer.
    - Rinse with 1 mL TST and add 3 mL freshly prepared ST buffer.
    - Collect 5 mL of lysate in a Falcon tube on ice.

### Step 4: Centrifugation (5m)
- Centrifuge at 500 x g for 5 minutes at 4°C in a swinging bucket centrifuge.

### Step 5: Pellet Resuspension
- Remove excess liquid with a P200 pipette carefully without disturbing the pellet.
- Gently resuspend the pellet with a P1000 pipette aiming to recover 6000 nuclei using a diluted nuclei buffer containing 1U/µL RNase inhibitor.

### Step 6: Nuclei Counting
- Use a C-chip disposable haemocytometer to count nuclei.
- Confirm counts and assess debris and nuclear membrane integrity.

### Step 7: Confirmation
- Confirm nuclei counts with a Bio-Rad TC20 for viable cells. Ideal live cell proportion: 1-4%.

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