Aug 13, 2025
Nuclei Isolation for 10x Multiome
- Nicholas Pease1
- 1University of Pittsburgh
Protocol Citation: Nicholas Pease 2025. Nuclei Isolation for 10x Multiome. protocols.io https://dx.doi.org/10.17504/protocols.io.261gek3mwg47/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: August 12, 2025
Last Modified: August 13, 2025
Protocol Integer ID: 224554
Keywords: cultured human lymphocytes for 10x multiome processing, nuclei isolation for 10x multiome protocol, 10x multiome processing, 10x multiome protocol, nuclei isolation, isolation of nuclei, cultured human lymphocyte, nuclei
Abstract
Protocol for isolation of nuclei from cultured human lymphocytes for 10x Multiome processing.
Guidelines
Dead Cell Removal
1. Resuspend cells in 1mL of DCR buffer, pass through 40uM filter and pellet at 300xg for 10min
2. Resuspend each sample in 100uL of DCR buffer
3. Add 5 uL of DCR (Annexin V) Cocktail to each sample
4. Add 5 uL of Biotin Selection Cocktail to each sample
5. Mix and incubate at RT for 3min
6. Vortex RapidSpheres for 30 seconds
7. Add 10 uL RapidSpheres to each sample, mix gently to disperse rapidspheres, add 2.4mL of DCR buffer and mix by gently pipetting up and down 2-3 times
8. Place tubes (without lid) into magnet and incubate at RT for 10 min*
9. Use pipette to transfer supernatant to a new 15 mL tube
10. Add 7.5mL of 2% FBS buffer and pellet at 300xg for 10min at 4C
Nuclei Isolation
1. Resuspend cells in 1mL of PBS + 0.04% PBS and transfer to 2mL tube
2. Pellet at 350xg for 8min at 4C
3. Resuspend in 1mL of PBS + 0.04% PBS and pellet again
4. Remove supernatant and add 100uL of chilled Lysis Buffer, pipette to mix 10x. and incubate on ice for 3min
5. Add 1mL of chilled Wash Buffer to lysed cells and pipette mix 5x
6. Pellet at 500xg for 5min at 4C
7. Remove supernatant and repeat wash two more times for total of 3 washes
8. Resuspend at 1e6 cells/mL in chilled Diluted Nuclei Buffer
9. Count and load cells according to Chromium Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics)
Materials
Dead Cell Removal
- Dead cell removal (DCR) buffer: PBS containing 2% fetal bovine serum (FBS) and 1 mM CaCl
- 2% FBS buffer: PBS containing 2% FBS
Nuclei Isolation
- Diluted Nuclei Buffer: 1X Nuclei Buffer, 1mM DTT, 1U/uL RNase inhibitor
- Wash Buffer: 10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20, 1mM DTT, 1U/uL RNase inhibitor
- Lysis Buffer: 10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20, 1mM DTT, 1U/uL RNase inhibitor, 0.1% IGEPAL, 0.01% Digitonin
Troubleshooting
Dead Cell Removal
Resuspend cells in 1mL of dead cell removal (DCR) buffer (PBS with 2% FBS and 1mM CaCl), pass through 40uM filter and pellet at 300xg for 10min
Resuspend each sample in 100uL of DCR buffer
Add 5 uL of DCR (Annexin V) Cocktail to each sample
Add 5 uL of Biotin Selection Cocktail to each sample
Mix and incubate at RT for 3min
Vortex RapidSpheres for 30 seconds
Add 10 uL RapidSpheres to each sample, mix gently to disperse rapidspheres, add 2.4mL of DCR buffer and mix by gently pipetting up and down 2-3 times
Place tubes (without lid) into magnet and incubate at RT for 10 min*
Use pipette to transfer supernatant to a new 15 mL tube
Add 7.5mL of 2% FBS buffer and pellet at 300xg for 10min at 4C
Nuclei Isolation
Resuspend cells in 1mL of PBS + 0.04% PBS and transfer to 2mL tube
Pellet at 350xg for 8min at 4C
Resuspend in 1mL of PBS + 0.04% PBS and pellet again
Remove supernatant and add 100uL of chilled Lysis Buffer, pipette to mix 10x. and incubate on ice for 3min
Add 1mL of chilled Wash Buffer to lysed cells and pipette mix 5x
Pellet at 500xg for 5min at 4C
Remove supernatant and repeat wash two more times for total of 3 washes
Resuspend at 16e6 cells/mL in chilled Diluted Nuclei Buffer
Count and load cells according to Chromium Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics)