Jul 02, 2026

Mouse Heart Nuclei Isolation for 10x Genomics Single-Cell snRNA-seq or Multiome V.3

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqxzrklk5/v3
  • 1The Jackson Laboratory;
  • 2The Jackson Lab;
  • 3Single Cell Biology Lab, The Jackson Laboratory, USA
Greggory A A Perry
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqxzrklk5/v3
Protocol Citation: Greggory A Perry, Sandra Daigle, Jessica Grassmann, Grace Frohock, Mike Hastings, William F Flynn, Elise Courtois 2026. Mouse Heart Nuclei Isolation for 10x Genomics Single-Cell snRNA-seq or Multiome. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqxzrklk5/v3Version created by Greggory A A Perry
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 01, 2026
Last Modified: July 02, 2026
Protocol  Integer ID: 320194
Keywords: mouse heart nuclei isolation, 10x genomics scrnaseq, frozen mouse heart tissue, downstream snrna, minimal debris for downstream snrna, multiome this sop, nuclei, seq assay, multiome, mouse heart nuclei isolation for 10x genomics single, 10x genomics single, snrna
Abstract
This SOP aims to extract nuclei from unfixed snap-frozen mouse heart tissue (~50mg). It was optimized and tested on both whole and pulverized tissue, with no differences noted between these. These preps resulted in minimal debris for downstream snRNA-seq assays on the 10x Genomics Chromium X.

This protocol enables processing up to 4 samples simultaneously with 1 person, or 8 with 2.
Guidelines
A. Use wide-bore pipette tips to avoid damage when handling tissue or nuclei, and pipette slowly.
B. Keep tissue on dry ice until ready to start gentleMACS.

C. We have found that the Miltenyi Anti-Nucleus beads not only provide a cleaner final suspension, but also maximize the targeted nuclei by eliminating "Viable" cells from the suspension. Our data also show that this greatly improves sequencing data quality. This method is recommended.


Hoechst/Syto RNA Select Staining Guidelines

Nuclear staining can be performed to ensure the nuclei are not blebbing and that the RNA is intact. Below are our guidelines.
1. Extracellular debris should be minimal after the nuclei have been passed through the sucrose column.
2. Extracellular debris will be present in a much greater quantity if a sucrose column is not used. Debris can interfere with downstream 10x operations by introducing clogs and background ambient RNA.
3. Nuclei should be intact, with minimal blebbing and clumping. Nuclei blebbing will make them more prone to clumping, which can cause clogs during loading of the 10x chip.
4. The Hoechst and Syto Select staining should be uniform throughout the nuclei, but the Syto staining usually has more variation.
If working with human tissue, follow all institutional rules and guidelines.
Materials
10X Genomics Nuclei Isolation Kit – 1000494 
Nuclei Extraction Buffer: 130-128-024 (Miltenyi) 
Miltenyi Anti-Nucleus Microbeads – 130-132-997 
Miltenyi LS Column – 130-042-401 
QuadroMACS Separator: 130-090-976 (Miltenyi)
MultiStand: 130-042-303 (Miltenyi)
pluriStrainer Mini 40 µm - 43-10040-60 
MACS SmartStrainer,100 µm - 930-098-469 (Miltenyi)
ROCHE Protector RNase Inhibitor – 3335402001 
Gibco DPBS (1X) – 14190144 
MACS BSA Stock Solution: 130-091-376 (Miltenyi) 
Lobind 1.5ml Microcentrifuge Tubes 
Lobind 2ml Microcentrifuge Tubes 
5ml Centrifuge Tubes 
15ml Centrifuge Tubes 
Refrigerated Swinging Bucket Centrifuge 
Refrigerated Fixed Angle centrifuge 
Vortex/Mixer 
Minifuge 
Appropriate PPE – gloves, lab coat, safety glasses. 


















Before start
Set up all tubes and prepare all buffers before removing the tissue from dry ice.
Dissociation
30m
Dissociation of Frozen Whole or Pulverized Mouse Heart Tissue

  1. If frozen tissue is not already in a 1.5 mL microcentrifuge tube, transfer to pre-chilled 1.5 mL centrifuge tubes on dry ice.
  2. Place the tubes on ice and add 150 µL of 10x Chromium Nuclei Isolation Lysis Buffer to each tube.
  3. With the pestle provided in the 10X Nuclei Isolation Kit, pestle each tissue 15 times. 
  4. Start a 15-minute timer, then add an additional 350 µL lysis buffer to each tube on ice and follow the instructions during the time ranges in steps 5-7.
  5. 0-5 minutes: Pestle an additional 20-40 times, or as needed.
  6. 5-10 minutes: Pipette lysate 10-15 times with a wide-bore, or regular-bore P1000 pipette. 
  7. 10-15 minutes: Pipette lysate 10-15 times with a regular-bore pipette. 
  8. When the timer is finished, immediately transfer the lysate to an assembled 5 o15 mL tube using the Miltenyi 100 µm SmartStrainer.
  9. Rinse the filter with 500 µL of Nuclei Wash/Resuspension Buffer, tap, and discard the filter.
  10. Immediately transfer 500 µL of the lysate to the assembled Nuclei Isolation Column and the 2 mL Collection Tube. 
  11. Centrifuge at 16,000 rcf at 4°C for 30 seconds in a fixed-angle centrifuge. 
  12. Repeat into the same Collection Tube with the remaining lysate.
  13. Discard column, mix by vortexing at speed 3 for 5 seconds. 
  14. Centrifuge at 500 rcf at 4°C for 3 minutes.
  15. Remove supernatant and suspend in 450 µL Nuclei Separation Buffer with RNase Inhibitor. 
  16. Mix 15-20 times with a wide-bore pipette and pass through a pre-wet 40 µm Filter.
  17. Count Nuclei using a stain to determine the count and viability with the Luna FX7. 

  • Diluting the nuclei 1:1 for this count can yield a more accurate count: 4.5 µL lysate, 4.5 µL Nuclei Separation Buffer, 1 µL AOPi (total of 10 µL).

30m
Miltenyi Anti-Nucleus Microbead Cleanup
30m
Miltenyi Anti-Nucleus Microbead Incubation, Wash, and Elution

Miltenyi Anti-Nucleus Microbead Incubation

  1. Distribute 106 nuclei per sample into separate 5 mL tubes and normalize the volume to 450 µL with Nuclei Separation Buffer containing RNase Inhibitor.
  2. Add 50 µL of Miltenyi Anti-Nucleus Beads per 106 nuclei; mix by vortexing at speed 3 for 5 seconds.
  3. Incubate at 4°C (not on ice) for 15 minutes. Start a timer. Do not exceed 15 minutes; non-specific binding to debris may occur.


  • You can take the % of viable nuclei into account and add more than 10^6, depending on the viability. i.e., If the viability is 30%, you can add 1.3 million nuclei to maximize the return without adding more Anti-Nucleus Microbeads.
20m
LS Column Loading and washing

  1. Place Miltenyi LS columns on the magnetic stand, with 15 mL tubes beneath to collect waste.
  2. When the timer has 3 minutes left, add 3 mL Nuclei Separation Buffer without RNase Inhibitor to each LS column. Using NSB without RNase inhibitor in this step is optional; it can save on reagents.
  3. Remove samples from 4°C and add an additional 2 mL Nuclei Separation Buffer with RNase Inhibitor. Mix by vortexing at speed 3 for 3 seconds.
  4. Add the samples to the labeled LS columns.
  5. Wash the LS column 3 times with 1 mL Nuclei Separation Buffer with RNase Inhibitor. Rinse the sidewalls to ensure all nuclei are washed to the magnet.
  6. Remove the LS Column from the magnet and place it in the collection rack with a 2 mL tube nested inside a 5 or 15 mL tube (We had a special rack 3D-printed to fit 5 mL tubes, but you can elute directly into a 15 mL tube).
  7. Elute with 1 mL Nuclei Separation Buffer with RNase Inhibitor directly in the center of the column to avoid washing any remaining debris from the sides through the column.
  8. Count Nuclei using a stain to determine the number and viability. The Viability should be zero or very close to zero.
  9. If nuclei need to be fixed, spin down the suspensions and add the fixation buffer directly to the pellet. Follow the current 10x Genomics revision of protocol CG000782.






10m
Final Suspension
10m
Final Suspension if moving directly to snRNA-seq or Multiome

  1. Use the previous count to estimate the volume of suspension buffer needed, depending on your downstream needs.
  2. Centrifuge at 500 rcf at 4°C for 5 minutes.
  3. Remove the supernatant, leaving up to 30 μL behind to avoid loss, and add the appropriate volume of Nuclei Suspension Buffer.
  4. Mix 15-20 times with a wide-bore pipette and pass through a pre-wet 40 µm Filter.
  5. Count Nuclei using a stain to determine the number and viability.


5m