Jul 01, 2025
Nuclei isolation from brain tissue for single-cell ATAC
- Koen Theunis1,2,3,4,
- Suresh Poovathingal5,6
- 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
- Aertslab
Protocol Citation: Koen Theunis, Suresh Poovathingal 2025. Nuclei isolation from brain tissue for single-cell ATAC. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnrb7ql5d/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: June 30, 2025
Last Modified: July 01, 2025
Protocol Integer ID: 221299
Keywords: ASAPCRN, nuclei isolation from brain tissue, cell atac this protocol, nuclei isolation, cell atac assay, nuclei from brain tissue, isolating nuclei, cell atac, brain tissue, atac, single cell, nuclei, isolation
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-025179
Abstract
This protocol is for isolating nuclei from brain tissue and prepare them for single-cell ATAC assays like the 10X Genomics Chromium Next GEM Single Cell ATAC Kit, HyDrop-ATAC, etc.
Attachments
Materials
| A | B | C | D | E | F | |
| 2X HB- (Homogenization Buffer) | Stock conc | Final conc | V (µl) | |||
| Sucrose | 2000 | mM | 640 | mM | 2245,6 | |
| NaCl | 5000 | mM | 20 | mM | 28,1 | |
| MgCl2 | 1000 | mM | 6 | mM | 42,1 | |
| Tris pH7,5 | 1000 | mM | 20 | mM | 140,4 | |
| EDTA | 500 | mM | 0,2 | mM | 2,8 | |
| BSA | 7,5 | % | 0,08 | % | 74,9 | |
| Tween20 | 10 | % | 0,02 | % | 14,0 | |
| Protienase inhibitor | 50 | X | 2 | X | 280,7 | |
| DTT | 1000 | mM | 2 | mM | 14,0 | |
| Water | 4175,0 | |||||
| Total Volume | 7017,6 |
| A | B | C | D | E | F | |
| 1X HB+ (Homogenization Buffer) | Stock conc | Final conc | V (µl) | |||
| 2X HB- | 2 | X | 1 | X | 2200,0 | |
| Igepal | 10 | % | 0,05 | % | 22,0 | |
| Digitonin | 2 | % | 0,0025 | % | 5,5 | |
| Water | 2178,0 | |||||
| Total Volume | 4400,0 |
| A | B | C | D | E | F | |
| 1X HB- (Homogenization Buffer) | Stock conc | Final conc | V (µl) | |||
| 2X HB- (Homogenization Buffer) | 2 | X | 1 | X | 3648,0 | |
| Water | 3648,0 | |||||
| Total Volume | 7296,0 |
| A | B | C | D | E | F | |
| GM (Gradient Medium) | Stock conc | Final conc | V(µl) | |||
| CaCl2 | 1000 | mM | 5 | mM | 12,5 | |
| Optiprep | 60 | % | 50 | % | 2080,0 | |
| MgCl2 | 1000 | mM | 3 | mM | 7,5 | |
| Tris pH 7.5 | 1000 | mM | 10 | mM | 25,0 | |
| BSA | 7,5 | % | 0,04 | % | 13,3 | |
| Protienase inhibitor | 50 | X | 0,2 | X | 10,0 | |
| DTT | 1000 | mM | 1 | mM | 2,5 | |
| Water | 345,3 | |||||
| Total Volume | 2496,0 |
| A | B | C | D | E | F | |
| ODM (Optiprep Diluent Medium) | Stock conc | Final conc | V(µl) | |||
| KCl | 3000 | mM | 150 | mM | 114,6 | |
| MgCl2 | 1000 | mM | 30 | mM | 68,7 | |
| Tris pH 8 | 1000 | mM | 60 | mM | 137,5 | |
| Sucrose | 2000 | mM | 250 | mM | 286,4 | |
| Water | 1684,3 | |||||
| Total Volume | 2291,5 |
| A | B | C | D | E | F | |
| 29% Cushion | Stock conc | Final conc | V(µl) | |||
| Optiprep | 60 | % | 29 | % | 1786,4 | |
| ODM | 1909,6 | |||||
| Total Volume | 3696,0 |
| A | B | C | D | E | F | |
| Resuspension Buffer | Stock conc | Final conc | V(µl) | |||
| 20X Nuclei buffer | 20 | X | 1 | X | 22 | |
| BSA | 7,5 | % | 1 | % | 58,7 | |
| Water | 359,3 | |||||
| Total Volume | 440 |
Troubleshooting
A. Homogenization
Cut brain piece on dry ice, then transfer immediately in homogenizer containing 1 mL of ice-cold HB+. If tissue is still frozen, leave it in HB for 3 min before starting homogenization to prevent the nuclei from breaking.
Homogenize tissue with 10 manual gentle strokes (pestle A) + 10 manual gentle strokes (pestle B).
Put homogenate through 70 µm cell strainer over a 50 mL falcon (or immediately a 2 mL protein lo-bind tube).
Wash homogenizer & strainer with 1 mL HB- to a final volume of 2 mL.
Transfer the contents from the 50 mL falcon to a 2 mL protein lo-bind tube.
Spin nuclei @500g for 5 min in a swinging bucket and remove supernatant.
Resuspend pellet with HB- to get a total volume of 520 µL and transfer to a clean DNA lo-bind tube.
Add 520 µL of Gradient medium (GM) to sample (Vf = 1040 µL).
B. Isolation by centrifugation
Layer 770 µL of 29% Cushion in a 2 mL DNA LoBind tube.
Layer the sample on top of cushion using a P1000, without disturbing the cushion.
Centrifuge at least 3,000 rcf (you can go as high as 9,000) in a swinging bucket, at 4°C for 20 minutes with brake off.
Remove supernatant, remove the lower supernatant with P200, leaving about max 30 µL.
C. Resuspension and counting
Disrupt nuclei on bottom by pipetting with P200. And bring X µL to a fresh 1.5 mL LoBind tube.
Add X µL of 1X Resuspension buffer to tube to retain extra nuclei, and add them to the 1.5 mL tube from C.1.
Spin 5 min @ 350-450xg followed by resuspension in 1X Diluted nuclei buffer. Optional filter and count.