Jul 15, 2025
Nuclei Preparation from Frozen Tissue for 10X Multiome Using MACS
This protocol is a draft, published without a DOI.
- Justin Buchanan1,
- Allen Wang1,
- Jacinta Lucero1,
- Jovina Djulamsah1,
- Qian Yang1,
- Emily Eisner1,
- Michael Miller1,
- Betsy Smoot1
- 1UCSD Center for Epigenomics
- UCSD Center for Epigenomics
Protocol Citation: Justin Buchanan, Allen Wang, Jacinta Lucero, Jovina Djulamsah, Qian Yang, Emily Eisner, Michael Miller, Betsy Smoot 2025. Nuclei Preparation from Frozen Tissue for 10X Multiome Using MACS. protocols.io https://protocols.io/view/nuclei-preparation-from-frozen-tissue-for-10x-mult-g4nxbyvfp
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: July 02, 2025
Last Modified: July 15, 2025
Protocol Integer ID: 221623
Keywords: nuclei preparation from frozen tissue, 10x multiome, frozen human lung tissue, nuclei preparation, chromatin accessibility, frozen human lung tissue at the center, macs this protocol detail, gene expression, nuclei for input, chromatin accessibility in the same same cell, frozen tissue, nuclei, using mac, gene
Abstract
This protocol details how to isolate nuclei for input for 10x Multiome to profile gene expression and chromatin accessibility in the same same cell. Current standard for snap-frozen human lung tissue at the Center.
Materials
Reagent stocks:
25X Roche cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (5056489001, Sigma): per instructions, disolve 1 tablet in 2 mL of molecular biology water. Store frozen at -20 °C when not used. Product information: https://www.sigmaaldrich.com/US/en/product/roche/coedtafro?srsltid=AfmBOooJHGgMZdgK6UZtDm9_nct9CbqKV0Oj6q4QygE3yeI16b4VWDVd
Buffers (prepare fresh day of):
MACS Buffer (3 mL at ~20-40 mg of sample + ~1 mL per ~30 mg of additional sample): 5 mM CaCl2 (R040, G-Biosciences); 2 mM EDTA (15575020, Thermo Fisher Scientific); 1X Roche cOMPLETE Protease Inhibitor, EDTA-Free (11873580001, Sigma); 3 mM Mg-acetate (M2545, Sigma); 10 mM Tris-HCl, pH=8 (15568025, Thermo Fisher Scientific); 0.6 mM DTT (D9779, Sigma); 1 U/μL Recombinant RNAsin (N2515, Promega) in molecular biology water (46000-CM, Corning)
Sort Buffer (at least 3 mL per sample): 1X Roche cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (5056489001, Sigma); 1 U/μL Recombinant RNasin (Ribonuclease Inhibitor, N2515, Promega); 1% Fatty acid-free BSA (7500804, Lampire Biological Laboratories) in PBS (21-040-CV, Corning)
Collection Buffer (200-500 μL per sample): 5 U/μL Recombinant RNasin (Ribonuclease Inhibitor, N2515, Promega); 5% Fatty acid-free BSA (7500804, Lampire Biological Laboratories) in PBS (21-040-CV, Corning)
Wash Buffer (1 mL per sample): 10 mM Tris-HCl, pH 7.5 (15567-027, Invitrogen); 10 mM NaCl (AM9760G, Invitrogen); 3 mM MgCl2 (AM9530G, Invitrogen); 1% Fatty acid-free BSA (7500804, Lampire Biological Laboratories); 0.10% Tween-20 (Sigma, P7949-100ML); 1 mM DTT (D9779, Sigma); 1X Roche cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (5056489001, Sigma); 1 U/μL Recombinant RNasin (Ribonuclease Inhibitor, N2515, Promega) in molecular biology water (46000-CM, Corning)
Lysis Buffer (200 μL per sample): 10 mM Tris-HCl, pH 7.5 (15567-027, Invitrogen); 10 mM NaCl (AM9760G, Invitrogen); 3 mM MgCl2 (AM9530G, Invitrogen); 1% Fatty acid-free BSA (7500804, Lampire Biological Laboratories); 0.010% Tween-20 (Sigma, P7949-100ML); 0.010% IGEPAL CA-630 (I8896-50ML, Sigma); 0.001% Digitonin (G9441, Promega); 1X Roche cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (5056489001, Sigma); 1 mM DTT (D9779, Sigma); 1 U/μL Recombinant RNasin (Ribonuclease Inhibitor, N2515, Promega) in Molecular biology water (46000-CM, Corning)
Nuclei Buffer (200 μL - 1 mL per sample): 20X Nuclei Buffer (PN 2000207, 10X Genomics); 1 U/μL Recombinant RNasin (Ribonuclease Inhibitor, N2515, Promega); 1 mM DTT (D9779, Sigma) in molecular biology water (46000-CM, Corning)
Other Consumables:
gentleMACS M tubeMiltenyi BiotecCatalog #130-093-236
7-AADFisher ScientificCatalog #A1310
Sony Cell Sorter μM ChipSonyCatalog #LE-B3001
100 μM CellTricsSysmexCatalog #04-0042-2328
50 μM CellTricsSysmexCatalog #04-0042-2327
30 μM CellTricsSysmexCatalog #04-0042-2316
40 μM FlowmiFisher ScientificCatalog #14100150
Equipment:
Equipment
gentleMACS Octo Dissociator
NAME
Miltenyi Biotec
BRAND
130-134-029
SKU
Equipment
Cell Sorter
NAME
Sony
BRAND
SH800S
SKU
Equipment
Centrifuge
NAME
Eppendorf
BRAND
5980 R
SKU
Troubleshooting
B. Nuclei Preparation
Prechill any tubes, buffers or tools. Set the centrifuge to 4 °C .
Prepare all buffers fresh. Use low-stick protein tubes (e.g. Eppendorf LoBind) for all sample preparation steps. Add RNase and protease inhibitors just prior to experiment start.
MACS buffer to use is based on amount of input tissue. In general, use at least 40 mg of snap-frozen tissue in 1 mL of MACS buffer, and increase buffer amount 1 mL/20 mg of tissue.
Aliquot MACS buffer into gentleMACS M tube and deposit sample pieces into tube. ~30s-1m should pass in the time between samples being added to the tube and the tubes being processed on the MACS homogenizer.
Homogenize sample using octoMACS dissociator and protein_01_01 protocol (time: 00:53sec, rpr: 2753) in cold room (4 °C ).
Briefly pulse centrifuge the gentleMACS M tube to collect solution at the bottom of the tube.
Transfer solution to a 100 μM Celltrix filter into a fresh 5 mL tube. Wash the MACS tube and filter with 1 mL of MACS buffer.
Transfer the filtered lysate to a 50 μM Celltrix into another fresh 5 mL tube. Wash the filter and previous lysate tube with 1 mL of MACS buffer.
Centrifuge nuclei (500 rcf, 5 min, 4 °C ).
Discard supernatant. (A small amount of supernatant can be left to avoid disturbing the pellet).
Resuspend the pellet in 300-500 μL of sort buffer with 1:100 dilution of 7-AAD and incubate for 30 minutes in dark.
Transfer nuclei suspension to a 1.5 mL tube. Use 1 mL of sort buffer to wash the 5 mL tube and add to nuclei suspension for a total of 1.3-1.5 mL.
Centrifuge nuclei (500 rcf, 5 min, 4 °C ).
Remove supernatant and resuspend pellet in 1 mL of sort buffer.
Centrifuge nuclei (500 rcf, 5 min, 4 °C ).
Resuspend nuclei in 400 μL of sort buffer. Pass the lysate through a 30 μM Celltrix into a 1.5 mL tube. Wash filter with an additional 400-500 μL of sort buffer.
Sort ~300,000 nuclei into a 1.5 mL tube containing 200 μL of collection buffer. Do not exceed sample pressure of 5 during sort.
Spin down the sorted nuclei in collection buffer (500 rcf, 5 min, 4 °C ). Remove the supernatant.
Resuspend pellet in 100 μL of Lysis Buffer (pipette mix 5X). Incubate on ice for 2 minutes.
Immediately add 900 μL of Wash Buffer and pipette mix 5X. Remove entire suspension (set pipette to >1000 μL) and gently pass through a 40 μM Flowmi filter (attaches to end of pipette tip while suspension is in pipette tip) into a fresh 1.5 mL Lobind tube.
Centrifuge nuclei (500 rcf, 5 min, 4 °C ).
Remove supernatant and resuspend in 1X Nuclei Buffer. Specific volumes vary by sample, but typically ~7-15 μL is used.
Prepare a 1:20 dilution of nuclei for counting using 1 μL of nuclei, 9 μL of Nuclei Buffer, and 10 μL of Trypan Blue.
Count on a hemocytometer and record images from the microscope field. Target concentration is 3,500-8,000 nuclei/μL. Dilute and recount as needed.
Load 15,300 nuclei per tagmentation reaction, following the 10x Genomics Single Cell Multiome ATAC + Gene Expression protocol for the remainder of the experiment.