Nuclei isolation of fresh frozen human brain samples for for scRNA-seq

Koen Theunis; Suresh Poovathingal; Sarah Geurs

Jan 13, 2026

Nuclei isolation of fresh frozen human brain samples for for scRNA-seq

DOI

https://dx.doi.org/10.17504/protocols.io.5qpvo1rddg4o/v1

Koen Theunis^1,2,3,4,
Suresh Poovathingal^5,6,
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

Koen Theunis

Laboratory of Computational Biology, VIB Center for AI & Com...

DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo1rddg4o/v1

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

SN-Isolation-forRNA_...

18KB

Materials

 
AB
HOW MANY SAMPLES TO PROCESS?2
Concentration of Sucrose?2 M
 
2X salt-Tris solution
ABCD
2X salt-Tris solutionStock concFinal concV (µl)
NaCl5000 mM292 mM1168.0
Tris pH7.51000 mM20 mM400.0
CaCl1000 mM2 mM40.0
MgCl21000 mM42 mM840.0
Water17552.0
Total Volume20000.0
  
TW20+ Homogenization buffer
ABCD
TW20+ Homogenization bufferStock concFinal concV (µl)
2X salt-Tris solution2 X1 X1100.0
Tween-20 10%0.03%6.6
BSA7.5%0.01%2.9
KCl3000 mM25 mM18.3
Sucrose2000 mM250 mM275.0
DTT*1000 mM1 mM2.2
cOmplete protease inhibitor (Roche EDTA free)* 50 X0.5 X22.0
RNasin Plus (Promega)*40 U/ul0.5 U/ul27.5
Water745.4
Total Volume2200.0
 
  TW20- Homogenization buffer
ABCD
TW20- Homogenization buffer Stock concFinal concV (µl)
2X salt-Tris solution2 X1 X572.0
BSA7.5%0.01%1.5
KCl3000 mM25 mM9.5
Sucrose2000 mM250 mM143.0
DTT*1000 mM1 mM1.1
cOmplete protease inhibitor (Roche EDTA free)* 50 X0.2 X4.6
RNasin Plus (Promega)*40 U/ul0.2 U/ul5.7
Water406.5
Total Volume1144.0
  
GM (Gradient Medium)
ABCD
GM (Gradient Medium)Stock conc Final concV(µl)
CaCl1000 mM1 mM1.1
Optiprep*60%50%953.3
MgCl21000 mM5 mM5.7
Tris pH 7.51000 mM10 mM11.4
Sucrose2000 mM75 mM42.9
DTT*1000 mM1 mM1.1
cOmplete protease inhibitor*50 X0.5 X11.4
RNasin Plus (Promega)*40 U/ul0.5 U/ul14.3
Water102.6
Total Volume1144
  
ODM (Optiprep Diluent Medium)
ABCD
ODM (Optiprep Diluent Medium)Stock conc Final concV(µl)
KCl3000 mM150 mM48.1
MgCl21000 mM30 mM28.9
Tris pH 81000 mM60 mM57.8
Sucrose2000 mM250 mM120.3
Water707.6
Total Volume963
  
29% Cushion
ABCD
29% CushionStock conc Final concV(µl)
Optiprep60%29%819
ODM875
Total Volume1694

Resuspension Buffer
ABCD
Resuspension BufferStock conc Final concV(µl)
PBS1 X1 X192.5
BSA7.5%0.75%22
RNasin Plus (Promega)*40 U/uL1 U/uL5.5
220
 *Add these components just before use.

A. Preparation

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.

B. Homogenization

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.

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.

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

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.

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

	A	B
	HOW MANY SAMPLES TO PROCESS?	2
	Concentration of Sucrose?	2 M

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

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

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

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

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

A	B	C	D
29% Cushion	Stock conc	Final conc	V(µl)
Optiprep	60%	29%	819
ODM			875
Total Volume			1694

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