Aug 14, 2025
Nuclei preparation from frozen tissue for Chromium Single Cell Multiome ATAC + Gene Expression (10x Genomics)
- Nathan Zemke1,
- Bing Ren1
- 1University of California, San Diego
Protocol Citation: Nathan Zemke, Bing Ren 2025. Nuclei preparation from frozen tissue for Chromium Single Cell Multiome ATAC + Gene Expression (10x Genomics). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx242gx1/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.
Tissue collection for this protocol needs prior approval by the users' Institutional Review Board (IRB) or equivalent ethics committee
Created: September 14, 2023
Last Modified: August 14, 2025
Protocol Integer ID: 91550
Keywords: chromium single cell multiome atac, nuclei preparation from frozen tissue, multiome atac, 10x genomic, chromium single cell, nuclei preparation, preparation from frozen tissue, tissue collection for this protocol, gene expression, frozen tissue, gene, tissue collection
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Abstract
This protocol details nuclei preparation from frozen tissue for Chromium Single Cell Multiome ATAC + Gene Expression (10x Genomics). Tissue collection for this protocol needs prior approval by the users' Institutional Review Board (IRB) or equivalent ethics committee
Attachments
841-276.pdf
212KB
Materials
Reagents and consumables
Note
Prepare buffers fresh and leave On ice .
NIM (Can be stored at 4 °C )
| A | B | C | D | |
| Reagent | Stock c | Final c | for 5 mL | |
| Sucrose (S1888, Sigma) | 1M | 0.25M | 1.25 mL | |
| KCl (AM9640G, Invitrogen) | 2M | 25 mM | 62.5 μl | |
| MgCl2 (194698, Mp Biomedicals Inc) | 1M | 5 mM | 25 μl | |
| Tris-HCl, pH 7.5 (15567027, Thermo Fischer Scientific) | 1M | 10 mM | 50 μl | |
| Molecular biology water (46000-CM, Corning) | - | - | 3.613 ml |
NIM-DP (Make fresh)
| A | B | C | D | |
| Reagent | Stock c | Final c | For 2 mL | |
| NIM buffer | 1X | 1X | 1.9 ml | |
| DTT (D9779, Sigma) | 200 mM | 1 mM | 10 μl | |
| Pierce Protease Inhibitor | 50X | 1X | 40 μl | |
| Recombinant RNAsin (Promega, PAN2515) | 40 U/μl | 1.0 U/μl | 50 μl |
NIM-DP-L
| A | B | C | D | |
| Reagent | Stock c | Final c | for 1 mL | |
| NIM-DP | 1X | 1X | 990 μl | |
| Triton X-100 (Sigma, T8787-100ML) | 10% (in water) | 0.10% | 10 μl |
Sort buffer (SB) (Can be stored at 4C without RNasin or protease inh)
| A | B | C | D | |
| Reagent | Stock c | Final c | 5 samples (500 uL each) | |
| EDTA (Invitrogen, 15575020) | 500 mM | 1 mM | 5 μl | |
| Recombinant RNAsin (Promega, PAN2515) | 40 U/ul | 1 U/ul | 62.5 μl | |
| Pierce Protease Inhibitor | 50X | 1X | 50 μl | |
| Fatty acid free BSA in PBS | 10% | 1% | 250 uL | |
| PBS | - | - | 2.133 mL |
Collection buffer (CB) (Can be stored at 4C without RNasin or protease inh)
| A | B | C | D | |
| Reagent | Stock c | Final c | 5 samples (87.5 uL each) | |
| Recombinant RNAsin (Promega, PAN2515) | 40 U/μl | 5 U/μl | 54.7 μl | |
| Pierce Protease Inhibitor | 50X | 1X | 8.75 μl | |
| Fatty acid free BSA in PBS | 10% | 5% | 218.75 μl | |
| PBS | - | - | 155.3 μl |
5X OMNI (Permeabilization buffer) (Make fresh)
| A | B | C | D | |
| Reagent | Stock c | Final c | for 200 μL | |
| Tris-HCl (pH 7.4) (15567027, Thermo Fischer Scientific) | 1M | 50 mM | 10 μL | |
| NaCl (Fischer, S271-3) | 5M | 50 mM | 2 μl | |
| MgCl2 (194698, Mp Biomedicals Inc) | 1M | 15 mM | 3 μl | |
| Tween-20 (Sigma, P7949-100ML) | 10% | 0.05% | 1 μl | |
| IGEPAL (Sigma, I8896) | 10% | 0.05% | 1 μl | |
| Digitonin (Promega, G9441) | 2% | 0.01% | 0.5 μl | |
| Fatty acid free BSA in PBS | 10% | 5% | 100 μl | |
| DTT (D9779, Sigma) | 200mM | 5mM | 5 μl | |
| Recombinant RNAsin (Promega, PAN2515) | 40 U/μl | 1U/μl | 5 μl | |
| Pierce Protease Inhibitor | 50X | 5X | 20 μl | |
| Molecular biology water (46000-CM, Corning) | - | - | 52.5 μl |
Wash Buffer (Make fresh)
| A | B | C | D | |
| Reagent | Stock c | Final c | For 2 mL | |
| Tris-HCl (pH 7.4) (15567027, ThermoFischer Scientific) | 1 M | 10 mM | 20 μL | |
| NaCl (Fischer, S271-3) | 5 M | 10 mM | 4 μl | |
| MgCl2 (194698, Mp Biomedicals Inc) | 1 M | 3 mM | 6 μl | |
| Tween-20 (Sigma, P7949-100ML) | 10% | 0.10% | 20 μl | |
| Fatty acid free BSA in PBS | 10% | 1% | 200 μl | |
| DTT (D9779, Sigma) | 200 mM | 1 mM | 10 μl | |
| Recombinant RNAsin (Promega, PAN2515) | 40 U/μl | 1 U/μl | 50 μl | |
| Pierce Protease Inhibitor | 50X | 1X | 40 μl | |
| Molecular biology water (46000-CM, Corning) | - | - | 1.65 ml |
1X Nuclei Buffer (Make fresh)
| A | B | C | D | |
| Reagent | Stock c | Final c | 100 uL | |
| 20X Nuclei Buffer (10X Genomics kit) | 20X | 1X | 5 μl | |
| DTT (D9779, Sigma) | 200 mM | 1 mM | 0.5 μl | |
| Recombinant RNAsin (Promega, PAN2515) | 40 U/μl | 1 U/μl | 2.5 μl | |
| Molecular biology water (46000-CM, Corning) | - | - | 92 μl |
- 7-AAD (Invitrogen, A1310)
- Sony Cell Sorter 100 μM Chip (Sony, LE-B3001)
- Sony Cell Sorter (Sony, SH800S)
- Eppendorf centrifuge (Eppendorf, 5920 R)
- 30 μm CellTrics (Sysmex, 04-0042-2316)
- 1 ml Dounce Tissue Grinder (Wheaton, 357538)
- 1.5 ml LoBind tubes (Eppendorf, 22431021)
SucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1888
2M KClInvitrogen - Thermo FisherCatalog #AM9640G
Magnesium chloride, hexahydrate, cell culture reagentMP BiomedicalsCatalog #02194698-CF
1M Tris-HCl pH 7.5Thermo Fisher ScientificCatalog #15567027
Molecular Biology Grade Water Tested to USP Sterile Purified Water SpecificationsCorningCatalog #46-000-CM
DL-Dithiothreitol for molecular biology ≥98% (HPLC) ≥99% (titration)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D9779
Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100ML
Invitrogen™ UltraPure™ 0.5M EDTA, pH 8.0Fisher ScientificCatalog #15-575-020
Sodium ChlorideFisher ScientificCatalog #S271-3
TWEEN 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
IgepalMerck MilliporeSigma (Sigma-Aldrich)Catalog #I8896
DigitoninPromegaCatalog #G9441
7-AAD (7-Aminoactinomycin D)Thermo FisherCatalog #A1310
WHEATON® Dounce Tissue Grinder, 1 mLDWK Life SciencesCatalog #357538
1.5 mL LoBind tubes EppendorfCatalog #022431021
Equipment
SH800S cell sorter
NAME
Sony
BRAND
SH800S
SKU
https://www.sonybiotechnology.com/us/instruments/sh800s-cell-sorter/specifications/
SPECIFICATIONS
Equipment
Centrifuge 5920 R
NAME
Eppendorf
BRAND
5920 R
SKU
LINK
Troubleshooting
Nuclei preparation
31m
Prechill any tubes, buffers or tools. Set the centrifuge to 4 °C .
For each sample to be homogenized, prepare a Dounce Tissue Grinder with 2 pestles (“Loose” and “Tight”). Remove from 70% ethanol storage, rinse with MilliQ water three times. Place mortars (buckets) in ice. Dry pestles with kimwipe and place on clean parafilm on top of ice to chill.
Remove excessive water collected at the bottom of mortars with p1000.
Add 990 µL of NIM-DP and 10 µL of Triton X-100 to each bucket to make 1 mL of NIM-DP-L. Pipette mix.
Tip tissue into mortar or resuspend tissue in 1 mL of NIM-DP-L buffer and transfer to a Dounce homogenizer.
Note
Be gentle and avoid introducing bubbles.
Homogenize the sample by using the Loose pestle (usually 5-10 strokes) followed by the tight pestle (usually 15-25). Switch pestles when most of the tissue has been broken up into small pieces and use the tight pestle to homogenize until the solution is uniform (no obvious particles).
Filter using a 30 µm CellTrics filter into LoBind tube.
Centrifuge nuclei 1000 rcf, 4°C, 00:10:00 .
10m
Discard the supernatant and gently resuspend pellet in 1 mL of NIM-DP. Centrifuge nuclei 1000 rcf, 4°C, 00:10:00 .
10m
Add 7-AAD to Sort Buffer (1:1000) to a final concentration of 2 micromolar (µM) .
Discard supernatant from pelleted nuclei. Gently resuspend pellet in 400 µL of sort buffer + 7-AAD by mixing with a p1000 pipette ~5 times or until no clumps are visible.
Sort 120,000 nuclei into a LoBind tube containing 87.5 µL of collection buffer.
Measure the total volume of the sorted nuclei in the collection buffer via reverse pipetting.
Add the appropriate amount of 5X permeabilization buffer for a final concentration of 1X, and gently pipette mix with p1000 5 times.
Incubate On ice for 00:01:00 , then centrifuge 500 rcf, 4°C, 00:05:00 .
6m
Slowly remove the supernatant until 20-30 μL remains in the tube.
Note
Do not disturb pellet.
Slowly add 650 µL of Wash Buffer along wall of tube (try not to disturb pellet) and immediately centrifuge 500 rcf, 4°C, 00:05:00 .
5m
Prepare tubes for counting nuclei on hemocytometer by adding 7 µL of 1X Nuclei Buffer to each tube for a 1:16 dilution.
Very carefully remove the supernatant, switching to a p20 pipette once the supernatant volume is <20 µL . Leave ~1 µL to avoid disturbing the pellet.
Add 7 µL of 1X Nuclei Buffer and very gently resuspend pellet 4 times. Immediately take 1 µL and add to tube for counting containing 7 µL of 1X Nuclei Buffer.
Count nuclei by stain with 8 µL Trypan Blue (Invitrogen, T10282), count on a hemocytometer and record images from the microscope field. For calculating nuclei concentration of original stock multiply count average x 10 x 16 (dilution factor).
Refer to Chromium Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics) Step 1. Load 18 - 20K nuclei per tagmentation.