Jan 13, 2026
Nuclei isolation of fresh frozen human brain samples for for scRNA-seq
- Koen Theunis1,2,3,4,
- Suresh Poovathingal5,6,
- Sarah Geurs7
- 1Laboratory of Computational Biology, VIB Center for AI & Computational Biology, Leuven, Belgium;
- 2VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium;
- 3Department of Human Genetics, KU Leuven, Leuven, Belgium;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, United States;
- 5VIB KU Leuven Center for Brain & Disease Research;
- 6VIB Center for AI & Computational Biology (VIB.AI), Leuven, Belgium;
- 7VIB Tech Watch, VIB Technologies, Ghent, Belgium
- Aertslab
Protocol Citation: Koen Theunis, Suresh Poovathingal, Sarah Geurs 2026. Nuclei isolation of fresh frozen human brain samples for for scRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo1rddg4o/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: September 09, 2025
Last Modified: January 13, 2026
Protocol Integer ID: 238288
Keywords: ASAPCRN, scRNA-seq, nuclei isolation, nuclei isolation for scrna, nuclei isolation of fresh frozen human brain sample, fresh frozen human brain sample, scrna, nuclei isolation, seq, nuclei
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000430
Aligning Science Across Parkinson’s
Grant ID: ASAP-025179
Abstract
This protocol describes nuclei isolation for scRNA-seq.
Attachments
Materials
| A | B | |
| HOW MANY SAMPLES TO PROCESS? | 2 | |
| Concentration of Sucrose? | 2 M |
2X salt-Tris solution
| A | B | C | D | |
| 2X salt-Tris solution | Stock conc | Final conc | V (µl) | |
| NaCl | 5000 mM | 292 mM | 1168.0 | |
| Tris pH7.5 | 1000 mM | 20 mM | 400.0 | |
| CaCl | 1000 mM | 2 mM | 40.0 | |
| MgCl2 | 1000 mM | 42 mM | 840.0 | |
| Water | 17552.0 | |||
| Total Volume | 20000.0 |
TW20+ Homogenization buffer
| A | B | C | D | |
| TW20+ Homogenization buffer | Stock conc | Final conc | V (µl) | |
| 2X salt-Tris solution | 2 X | 1 X | 1100.0 | |
| Tween-20 | 10% | 0.03% | 6.6 | |
| BSA | 7.5% | 0.01% | 2.9 | |
| KCl | 3000 mM | 25 mM | 18.3 | |
| Sucrose | 2000 mM | 250 mM | 275.0 | |
| DTT* | 1000 mM | 1 mM | 2.2 | |
| cOmplete protease inhibitor (Roche EDTA free)* | 50 X | 0.5 X | 22.0 | |
| RNasin Plus (Promega)* | 40 U/ul | 0.5 U/ul | 27.5 | |
| Water | 745.4 | |||
| Total Volume | 2200.0 |
TW20- Homogenization buffer
| A | B | C | D | |
| TW20- Homogenization buffer | Stock conc | Final conc | V (µl) | |
| 2X salt-Tris solution | 2 X | 1 X | 572.0 | |
| BSA | 7.5% | 0.01% | 1.5 | |
| KCl | 3000 mM | 25 mM | 9.5 | |
| Sucrose | 2000 mM | 250 mM | 143.0 | |
| DTT* | 1000 mM | 1 mM | 1.1 | |
| cOmplete protease inhibitor (Roche EDTA free)* | 50 X | 0.2 X | 4.6 | |
| RNasin Plus (Promega)* | 40 U/ul | 0.2 U/ul | 5.7 | |
| Water | 406.5 | |||
| Total Volume | 1144.0 |
GM (Gradient Medium)
| A | B | C | D | |
| GM (Gradient Medium) | Stock conc | Final conc | V(µl) | |
| CaCl | 1000 mM | 1 mM | 1.1 | |
| Optiprep* | 60% | 50% | 953.3 | |
| MgCl2 | 1000 mM | 5 mM | 5.7 | |
| Tris pH 7.5 | 1000 mM | 10 mM | 11.4 | |
| Sucrose | 2000 mM | 75 mM | 42.9 | |
| DTT* | 1000 mM | 1 mM | 1.1 | |
| cOmplete protease inhibitor* | 50 X | 0.5 X | 11.4 | |
| RNasin Plus (Promega)* | 40 U/ul | 0.5 U/ul | 14.3 | |
| Water | 102.6 | |||
| Total Volume | 1144 |
ODM (Optiprep Diluent Medium)
| A | B | C | D | |
| ODM (Optiprep Diluent Medium) | Stock conc | Final conc | V(µl) | |
| KCl | 3000 mM | 150 mM | 48.1 | |
| MgCl2 | 1000 mM | 30 mM | 28.9 | |
| Tris pH 8 | 1000 mM | 60 mM | 57.8 | |
| Sucrose | 2000 mM | 250 mM | 120.3 | |
| Water | 707.6 | |||
| Total Volume | 963 |
29% Cushion
| A | B | C | D | |
| 29% Cushion | Stock conc | Final conc | V(µl) | |
| Optiprep | 60% | 29% | 819 | |
| ODM | 875 | |||
| Total Volume | 1694 |
Resuspension Buffer
| A | B | C | D | |
| Resuspension Buffer | Stock conc | Final conc | V(µl) | |
| PBS | 1 X | 1 X | 192.5 | |
| BSA | 7.5% | 0.75% | 22 | |
| RNasin Plus (Promega)* | 40 U/uL | 1 U/uL | 5.5 | |
| 220 |
*Add these components just before use.
Troubleshooting
A. Preparation
5m
Prepare the 2X salt-Tris solution and aliquot to 1 ml tube and freeze at -20 °C .
Put the centrifuge for 4 °C .
Prepare the tween-20 lysis buffer fresh for each preparations.
Prepare cOmplete protease inhibitor (50X) by dissolving a tablet in 1 mL of water.
Filter sucrose solution and Protease inhibitor through 0.2 um/0.45 um filter.
Thaw all the reagents and prepare for the buffer reagents.
Place the homogenizer at -70 °C at the start of the experiment and place On ice 00:05:00 before homogenization.
5m
B. Homogenization
8m
Cut brain piece on dry ice (if large; > 20 mg -50 mg tissue).
Transfer 750 µL of ice-cold TW20+ HBuffer and tissue pieces to a homogenizer and leave it in +HB for 00:03:00 before starting homogenization.
3m
Homogenize tissue with 10 gentle strokes with pestle A, and 10 gentle strokes with pestle B.
Put homogenate through 70 µm cell strainer, wash homogenizer and filter with 250 µL TW+HB and leave the filterate On ice . Start the timer for 5 mins.
Transfer the filterate to a 2 mL protein low bind eppendorf and centrifuge @ 400 x g -500 x g for 00:05:00 . Discard the supernatant.
5m
Resuspend the pellet gently in 200 µL of TW20- HB, and transfer to 2 mL low-bind tube.
Add additional TW20- HB to reach a final volume of 520 µL .
Add 520 µL of Gradient Medium (Vf = 1040 µL ).
C. Isolation by centrifugation
20m
Layer 770 µL of 29% Cushion in the ultracentrifuge tube.
Layer the sample on top of cushion using a P1000, without disturbing the cushion.
Check tubes weight, adjust weight with HB/GM.
Centrifuge at least 3000 rcf with a swinging bucket rotor, at 4 °C for 00:20:00 with brake off.
20m
Remove supernatant, remove the lower supernatant with P200 or less, leaving around 50 µL -100 µL .
D. Resuspension and counting
5m
Disrupt nuclei on bottom by pipetting with P200. And bring total volume to a fresh 1.5 mL LoBind tube.
Add 100 µL of 1X Resuspension buffer to ultracentrifuge tube to retain extra nuclei, and add them to the 1.5 mL tube.
Spin 00:05:00 @ 350 x g -450 x g followed by resuspension in Resuspension buffer. Optional filter and count.
5m
Protocol references
This protocol was based on earlier work from others but with in-house modifications:
Marine Denechaud, Sarah Geurs, Thomas Comptdaer, Séverine Bégard, Alejandro Garcia-Núñez, Louis-Adrien Pechereau, Thomas Bouillet, Yannick Vermeiren, Peter P. De Deyn, Romain Perbet, Vincent Deramecourt, Claude-Alain Maurage, Michiel Vanderhaegen, Sebastiaan Vanuytven, Bruno Lefebvre, Elke Bogaert, Nicole Déglon, Thierry Voet, Morvane Colin, Luc Buée, Bart Dermaut, Marie-Christine Galas.
Tau promotes oxidative stress-associated cycling neurons in S phase as a pro-survival mechanism: Possible implication for Alzheimer’s disease.
Progress in Neurobiology, Volume 223, 2023, 102386, ISSN 0301-0082, https://doi.org/10.1016/j.pneurobio.2022.102386.
L. Swiech, M. Heidenreich, A. Banerjee, N. Habib, Y. Li, J. Trombetta, M. Sur, F. Zhang
In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9
Nat. Biotechnol., 33 (2015), pp. 102-106, 10.1038/nbt.3055