Jul 08, 2025
Nuclei isolation from fresh frozen tissues for 10X Single Cell Multiome ATAC + Gene Expression Sequencing and other technologies
- Amy Guillaumet-Adkins1,
- Amrita Sule1
- 1Broad Institute
- dGTEx Single Cell Protocols
Protocol Citation: Amy Guillaumet-Adkins, Amrita Sule 2025. Nuclei isolation from fresh frozen tissues for 10X Single Cell Multiome ATAC + Gene Expression Sequencing and other technologies. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3q7dqv25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2025
Last Modified: July 10, 2025
Protocol Integer ID: 117944
Keywords: 10x single cell multiome atac, gene expression sequencing, scale biosciences rna, sequencing data, nuclei from fresh frozen tissue sample, nuclei isolation from fresh frozen tissue, including rna metric, isolating nuclei, rna metrics such as gene, atac metrics like tss enrichment ratio, cell rna, nuclei isolation, rna, fresh frozen tissue sample, fresh frozen tissue, atac metric, umis per cell, gene expression, single cell, atac
Funders Acknowledgements:
Chan Zuckerberg Initiative (CZI) Seed Networks
Grant ID: CZF2019-002455
NIH
Grant ID: U24HG012090
Abstract
This protocol is designed for isolating nuclei from fresh frozen tissue samples ranging from 6 mg to 30 mg. It has been primarily used to generate 10X Single Cell Multiome ATAC + Gene Expression sequencing data across 21 different tissue types (see the guidelines and warnings section for details). For some tissues, debris removal is performed using a continuous density gradient—following the Daughter of Frankenstein protocol—instead of FACS sorting.
The protocol consistently delivers strong quality control (QC) metrics, including RNA metrics such as genes per cell and UMIs per cell, and ATAC metrics like TSS enrichment ratio and high-quality fragment fractions. Beyond 10X 3' v3.1 single-cell RNA sequencing, nuclei isolated using this method have also been successfully applied to other single-cell platforms, such as Fluent BioSciences PIPseq and Scale Biosciences RNA sequencing. This protocol is compatible with a wide range of downstream technologies.
Guidelines
21 Fresh frozen tissues optimized by this protocol
List of fresh frozen tissues for which the 10X Multiome data was generated using this protocol. Density gradient
centrifugation is only necessary for certain tissues. Protocol deviation for aorta and pancreas are also listed in the
table.
Note: Before applying this protocol to a new tissue type, optimize it using the provided buffers (without RNase inhibitors). After completing Section I, assess debris by staining the nuclei with Propidium Iodide. If significant debris is observed, proceed to the density gradient centrifugation step; if not, proceed directly to Section III.
Materials
Reagents:
Nuclei Buffer 20X 10x GenomicsCatalog #2000153/2000207 Digitonin (5%)Thermo FisherCatalog #BN2006 Trizma Hydrochloride Solution pH 7.4 Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2194 Sodium Chloride Solution 5 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #59222C Magnesium Chloride Solution 1 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028 Nonidet P40 SubstituteMerck MilliporeSigma (Sigma-Aldrich)Catalog # 74385 Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
MACS BSA Stock SolutionMiltenyi BiotecCatalog # 130-091-376
RNase-Free Disposable Pellet Pestles, With tubeThermo FisherCatalog #12141368 MACS SmartStrainers (70 µm)Miltenyi BiotecCatalog #130-098-462 10% Tween 20Bio-Rad LaboratoriesCatalog #1662404
Sterile single-pack CellTrics™ filters 30 uMSysmexCatalog #04-004-2326
RNaseZap®Thermo ScientificCatalog #AM9780
Noyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12
Bel-Art® Disposable PestlesMerck MilliporeSigma (Sigma-Aldrich)Catalog #BAF199230001-100EA
Filter, CellTrics ,5um,30um Diam,St(50/BX)Sysmex
Optiprep ( Iodixanol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D1556-250ML
INCYTO C-Chip™ Disposable HemacytometersVWR International (Avantor)Catalog #82030-468
NP-40 nuclei Lysis buffer: (1.2 mL/sample)- Prepare fresh and keep on ice
| A | B | C | D | E | |
| Stock | Final | For 2 mL (uL) | For 4 mL (uL) | ||
| Tris-HCl (pH 7.4) | 1 M | 10 mM | 20 | 40 | |
| NaCl | 5 M | 10 mM | 4 | 8 | |
| MgCl2 | 1 M | 3 mM | 6 | 12 | |
| Nonidet P40** | 10% | 0.10% | 20 | 40 | |
| DTT | 1000 mM | 1 mM | 2 | 4 | |
| RNase inhibitor 40 U/µl | 40 U/µl | 1 U/µl | 50 | 100 | |
| Nuclease-free Water | 1898 | 3796 |
PBS + 2% BSA + 1U/µl RNase I: (3.5 mL/sample)-Prepare fresh and keep on ice
| A | B | C | D | E | |
| Stock | Final | For 4 mL (uL) | For 8 mL (uL) | ||
| PBS | 1 M | 10 mM | 2700 | 5400 | |
| BSA | 10% | 1% | 800 | 1600 | |
| RNase inhibitor 40 U/µl | 40 U/µl | 1 U/µl | 100 | 200 |
Wash Buffer: (1 mL/sample)-Prepare fresh and keep on ice
| A | B | C | D | E | |
| Stock | Final | For 1 mL (uL) | For 2 mL (uL) | ||
| Tris-HCl (pH 7.4) | 1 M | 10 mM | 10 | 20 | |
| NaCl | 5 M | 10 mM | 2 | 4 | |
| MgCl2 | 1 M | 3 mM | 3 | 6 | |
| BSA | 10% | 1% | 100 | 200 | |
| Tween-20 | 10% | 0.10% | 10 | 20 | |
| DTT | 1000 mM | 1 mM | 2 | 1 | |
| RNase inhibitor | 40 U/µl | 1 U/µl | 25 | 50 | |
| Nuclease-free Water | 849 | 1698 |
1X Lysis Buffer: (250uL/sample) – Make at least 500 uL -Prepare fresh and keep on ice
| A | B | C | D | E | |
| Stock | Final | For 2 mL (uL) | For 0.5 (uL) | ||
| Tris-HCl (pH 7.4) | 1 M | 10 mM | 20 | 5 | |
| NaCl | 5 M | 10 mM | 4 | 1 | |
| MgCl2 | 1 M | 3 mM | 6 | 1.5 | |
| Tween-20 | 10% | 0.10% | 20 | 5 | |
| Nonidet P40** | 10% | 0.10% | 20 | 5 | |
| Digitonin* | 5% | 0.01 | 4 | 1 | |
| BSA | 10% | 1% | 200 | 50 | |
| DTT | 1000 mM | 1 mM | 2 | 0.5 | |
| RNase inhibitor 40 U/µl | 40 U/µl | 1 U/µl | 50 | 12.5 | |
| Nuclease-free Water | 1674 | 418.5 |
Lysis Dilution buffer: (100 uL/sample) Make at least 500 uL-Prepare fresh and keep on ice
| A | B | C | D | E | |
| Stock | Final | For 2 mL (uL) | For 1 mL (uL) | ||
| Tris-HCl (pH 7.4) | 1 M | 10 mM | 20 | 10 | |
| NaCl | 5 M | 10 mM | 4 | 2 | |
| MgCl2 | 1 M | 3 mM | 6 | 3 | |
| BSA | 10% | 1% | 200 | 100 | |
| DTT | 1000 mM | 1 mM | 2 | 2 | |
| RNase inhibitor 40 U/µl | 40 U/µl | 1 U/µl | 50 | 25 | |
| Nuclease-free Water | 1718 | 858 |
0.1X Lysis buffer: (100uL/sample)-Keep on ice
| A | B | C | D | E | |
| Stock | Final | For 2 mL (uL) | For 0.5 mL (uL) | ||
| 1X Lysis Buffer | 1X | 0.1X | 200 | 50 | |
| Lysis Dilution Buffer | 1800 | 450 |
Diluted Nuclei Buffer
| A | B | C | |
| For 0.5 mL (uL) | For 0.25 mL (uL) | ||
| 20 X nuclei buffer | 25 | 12.5 | |
| DTT | 0.5 | 0.25 | |
| RNAseI | 12.5 | 6.25 | |
| diH2O | 462.5 | 231.25 |
BUFFERS TO MAKE IN ADVANCE FOR DENSITY GRADIENT (Filter Sterilize GD and GH)
| A | B | C | |
| Stock Solutions | grams | mL diH2O | |
| 500 mM Tricine | 4.48 | 50 | |
| 1M KCl | 3.725 | 50 | |
| 1 M MgCl2 X 6 H2O | 10.15 | 50 |
For 2 Samples: (Add RNAse Inhibitors at 1U/uL)
| G60 | GD | GW | GH | ||
| GW | 3 mL | 0.6 mL | - | - | |
| G30 | - | - | 3 mL | 2 mL |
Troubleshooting
Safety warnings
Materials Guidelines
- Use low-bind plastics (e.g., 1.5 mL tubes, P200, and P1000 tips) as recommended by 10x Genomics.
- Utilize a swinging-bucket centrifuge for optimal nuclei yield.
Additional Recommendations
- Maintain an RNase-free environment throughout the procedure.
- Keep nuclei on ice at all times to preserve integrity.
- Minimize protocol duration to improve data quality.
Before start
Previous Day Preparation
- Clean forceps and scissors with RNaseZap.
- Wrap the cleaned instruments in aluminum foil and store them at -20°C overnight in preparation for the experiment.
Day of the Experiment PreparationRNase-Free Workspace
- Wipe down the working bench and all pipettes with RNaseZap to maintain an RNase-free environment.
Equipment Setup
- Set the centrifuge to 4°C.
- Preheat the thermomixer to 65°C to thaw Digitonin.
Required Plastics per Sample
- 70 µm strainers for 15 mL conical tubes (×1)
- 30 µm CellTrics strainers
- 15 mL conical tubes (×1)
- 1.5 mL low-bind, RNase-free microfuge tubes (×3)
Ice and Dry Ice Preparation
- Prepare an ice bucket for buffers and tissue chopping.
- Have dry ice ready to retrieve tissue from the -80°C freezer.
- Set up the 70 µm filter on a 15 mL conical tube and place it on ice, ready for use just before starting.
Section I :Tissue Homogenization
Do not thaw the tissue before lysis; keep it on dry ice at all times.
Weigh the tissue to confirm the desired input weight, ensuring it remains frozen throughout the process.
If tissue is not already in a 1.5 mL tube, transfer to a 1.5 mL tube.
After weighing the tissue, proceed to Section II.
Section II: Tissue Homogenization
31m
Add 400 µL of NP-40 lysis buffer to the tissue on wet ice.
Using forceps and scissors, chop the tissue 3 to 4 times in 400 µL NP-40 lysis buffer while keeping it on ice.
Note: Over-chopping can lead to debris.
After chopping, homogenize the sample using a pellet pestle in 400 µL of NP-40 lysis buffer on ice for 00:02:00 to maximum of 00:04:00 .
6m
Keep the tube on ice at all times, only lifting it briefly to check the progress of homogenization.
Note: Homogenizing in lysis buffer for longer than 5 minutes may negatively affect ATAC data quality.
Once homogenization is complete, add 800 µL of NP-40 lysis buffer to the tube.
Mix by pipetting up and down twice using a wide-bore pipette tip (a regular-bore tip may be used if the tissue disintegrates easily).
Pass the suspension through a 70 µm strainer into a 15 mL conical tube kept on wet ice.
Be sure to collect the flowthrough from the bottom of the filter, avoiding contact with the base of the strainer to prevent disturbing any retained debris.
Collect all the flowthrough and transfer it into a new 1.5 mL tube.
Centrifuge at 500 x g, 4°C, 00:10:00
10m
Carefully remove and discard the supernatant, leaving approximately 50 µL behind. Avoid disturbing the pellet.
Add 1 mL PBS + 1% BSA + 1U/µl RNase Inhibitor. DO NOT MIX.
Incubate for 00:05:00 on ice.
5m
Pipette mix to re-suspend the pellet.
Strain with 30 µm Celltrix strainer into a new 1.5 mL microfuge tube on ice.
Centrifuge at 500 x g, 4°C, 00:10:00 .
10m
If performing density gradient centrifugation, Make the GW AND G30 buffers in this step.
Note: If you're optimizing the protocol to determine whether density gradient centrifugation is necessary, examine the nuclei under a microscope after the next step, using propidium iodide staining.
After centrifugation, carefully remove as much of the supernatant as possible without disturbing the pellet. If the pellet appears loose, leave approximately 50 µL behind.
If your nuclei preparation contains a high amount of debris, perform density gradient centrifugation.
If proceeding with density gradient centrifugation, continue to Section II. Otherwise, skip ahead to Section III, step 35.
Only perform the steps in Section III if your downstream assay involves profiling chromatin accessibility using a single-cell ATAC assay. Otherwise, proceed with your chosen single-cell assay after completing Section I or Section II, depending on the quality of the nuclei.
Nuclei images
- Nuclei with debris
Nuclei from Lung before density gradient 20X EVOS M7000 Imaging System
Section II : Density Gradient Centrifugation
30m
Add 300 µL of G30 buffer to the pellet. Mix well but gently to re-suspend the pellet.
Carefully underlay 1 mL of the G30 buffer beneath the sample homogenate from the previous step. A clear separation between the homogenate and the G30 buffer should form—do not disturb this interface.
Centrifuge the nuclei at 8000 x g, 4°C, 00:20:00 .
20m
After centrifugation, the nuclei will form a pellet at the bottom, while contaminating membranes will float at the top.
After centrifugation, carefully remove the supernatant and add 1 mL of ice-cold PBS containing 1% BSA and 1 U/µL RNase inhibitor, without re-suspending the nuclei.
Centrifuge the nuclei at 500 x g, 4°C, 00:05:00 .
5m
Remove as much of the supernatant as possible without disturbing the pellet. If the pellet appears loose, leave approximately 50 µL behind. Then, re-suspend the pellet in 1 mL of PBS + 1% BSA + 1U/µl RNase inhibitor.
Centrifuge the nuclei at 500 x g, 00:05:00
5m
Remove the supernatant and re-suspend the pellet in 50 µL of ice-cold PBS + 2% BSA + 1U/µl RNase inhibitor.
If optimizing the nuclei isolation protocol, proceed with nuclei imaging and counting to evaluate yield and quality.
Nuclei image after density gradient
Lung nuclei after density gradient 20X EVOS M7000 Imaging System
Section III : Permeabilization
12m
Add 500 µL PBS + 1% BSA + 1U/µl RNase inhibitor to the pellet.
Centrifuge at 500 x g, 5°C, 00:05:00
5m
Remove the supernatant without disrupting the nuclei pellet.
Re-suspend the pellet in 100 µL 0.1X Lysis Buffer and pipette mix 5 times.
Incubate for 00:02:00 on ice.
2m
Add 1 mL Wash buffer and pipette mix 5 times.
Centrifuge at 500 x g, 00:05:00 .
5m
Carefully remove the supernatant without disturbing the nuclei pellet.
Re-suspend the pellet in nuclei suspension buffer appropriate for the chosen downstream single cell technology.
Count the nuclei after staining them with a dye such as propidium iodide.
Proceed with 10X Single Cell Multiome ATAC + Gene Expression Sequencing, or continue with your chosen assay.
Protocol references
1. Daughter of Frankenstein protocol for nuclei isolation from fresh and frozen tissues using OptiPrep‱ continuous gradient V.2 dx.doi.org/10.17504/protocols.io.bs99nh96
2. Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing