May 12, 2026
Single-Nucleus Microglial Enrichment from Frozen Brain Tissue
- Dominika Dostalova1,2,
- Pavel Abaffy1,3,
- Lukas Valihrach1,3
- 1Laboratory of Glial Biology and Omics Technologies, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic;
- 2Faculty of Science, Charles University, Prague, Czech Republic;
- 3GeneCore Facility, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic
Protocol Citation: Dominika Dostalova, Pavel Abaffy, Lukas Valihrach 2026. Single-Nucleus Microglial Enrichment from Frozen Brain Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4zemovo5/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: March 25, 2025
Last Modified: May 12, 2026
Protocol Integer ID: 124989
Keywords: Microglia, snRNA-seq, PU.1, Smart-seq3xpress, frozen tissue, enrichment, transcriptomics, selective microglial transcriptomic profiling from frozen brain tissue, nucleus microglial enrichment from frozen brain tissue, selective microglial transcriptomic profiling, resolution transcriptomic profiling of enriched microglial population, nucleus microglial enrichment, microglial nuclei, enriched microglial population, frozen brain tissue, transcriptomic profiling, maintaining rna quality, seq3xpress, molecular neurobiology, rna quality
Funders Acknowledgements:
Czech Science Foundation
Grant ID: 23-05327S
Czech Science Foundation
Grant ID: 24-11364S
Czech Science Foundation
Grant ID: 25-16979S
Ministry of Education, Youth, and Sports
Grant ID: MULTIOMICS_CZ CZ.02.01.01/00/23_020/0008540
Institute of Biotechnology of the Czech Academy of Sciences
Grant ID: RVO 86652036
Charles University, project GA UK
Grant ID: 2224
Abstract
We present a protocol for the selective isolation of microglial nuclei from fresh-frozen brain tissue using the transcription factor PU.1 as a nuclear marker. To ensure reliable PU.1 detection, the procedure includes a brief formaldehyde fixation step that preserves nuclear integrity while maintaining RNA quality. The method is fully compatible with Smart-seq3xpress, enabling a high-resolution transcriptomic profiling of enriched microglial populations. This protocol is associated with the article: "Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue", published in Cellular and Molecular Neurobiology, DOI: 10.1007/s10571-026-01743-5.
Materials
Reagents:
Dulbecco’s Phosphate Buffered SalineMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8537
Formaldehyde 36-38%Penta ChemicalsCatalog #14150-11000
GlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #50046
UltraPure DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977035
SucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #84097
Trizma Hydrochloride Solution pH 7.4 Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2194
5 M Sodium chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5150-1L
Magnesium Chloride Solution 1 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Nonidet P40 SubstituteMerck MilliporeSigma (Sigma-Aldrich)Catalog # 74385
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Bovine Albumin Fraction V (7.5% solution)Gibco - Thermo Fisher ScientificCatalog #15260037
1 x TE BufferThermo Fisher ScientificCatalog #12090015
dNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0182
SIRV-Set 4LexogenCatalog #141.01
Silicone Oil 100 cStMerck MilliporeSigma (Sigma-Aldrich)Catalog #378364-250ML
40% Polyethylene Glycol MW 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1458
Thermolabile Proteinase KNew England BiolabsCatalog #P8111S
SEQURNA™ Thermostable RNase inhibitorSEQURNA ABCatalog #SQ00203
Antibodies:
| Ab - marker | Ord No | Fluorochrome | Producer | |
| ACSA-2 | 130-116-245 | APC | Milteneyi | |
| NeuN | Mab377X | AF-488 | Merck | |
| PU.1 | 81886S | PE | CST |
Oligos:
| A | B | C | D | E | |
| OligodTVN30 | IDT | RNase-Free HPLC | 100/10uM | /5BiosG/ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN |
Plastics and other materials:
Millex-GP Syringe Filter Unit 0.22 µm polyethersulfone 33 mm gamma sterilizedMerck Millipore (EMD Millipore)Catalog #SLGP033RS
10 mL LL SyringeB. Braun SECatalog #4617100V
RNase-Free Disposable Pellet Pestles, Without tubeThermo FisherCatalog #12141364
CellTrics Filters (30um)SysmexCatalog #04-004-2326
Hard-Shell® 384-Well PCR Plates thin wall skirtedBio-Rad LaboratoriesCatalog #HSP3801
Kimble™ Kontes™ Dounce Tissue GrindersThermo FisherCatalog #K8853000002
Before start
1) Cool the centrifuge to 2 °C , along with the necessary rotors.
2) Cool CellTrics filters in the fridge.
3) Keep working surface clean and RNases-free.
4) Keep all solutions On ice and keep samples on dry ice.
5) Use the pipette tips with filters.
Solutions
Prepare following solutions On ice :
Fixation Solution (per sample):
| Reagent | Volume [µL] | C_stock | C_final | |
| DPBS -/- | 2432.5 | - | - | |
| Formaldehyde | 67.5 | 36% | 4% | |
| Total | 2500 |
2.5M Glycine (stock):
| Reagent | Volume | C_stock | C_final | |
| Glycine | 2.063 g | - | 2.5M | |
| Nuclease Free Water | 11 mL | - | - | |
| Total | 11 mL |
Weigh the Glycine in a tube and dilute in ~90% of the NFW. After the dilution, fill with NFW to the final volume.
2M Sucrose Solution (stock):
| Reagent | Volume | C_stock | C_final | |
| Sucrose | 6.846 g | - | 2M | |
| Tris-HCl pH 7,4 | 100 µL | 1M | 10mM | |
| NaCl | 20 µL | 5M | 10mM | |
| MgCl2 | 30 µL | 1M | 3mM | |
| Nuclease Free Water | fill up to 10 mL | - | - | |
| Total | 10 mL |
First dissolve the weighed Sucrose in ~65% of the final NFW volume, then add the other solutions and finally fill to the final volume with the remaining NFW. Once prepared, filter the Sucrose solution through a 0.22µm filter using a syringe with a Luer Lock.
Lysis Buffer (per sample):
| Reagent | Volume [µL] | C_stock | C_final | |
| Nuclease Free Water | 3260 | - | - | |
| Tris-HCl pH 7,4 | 40 | 1M | 10mM | |
| NaCl | 8 | 5M | 10mM | |
| MgCl2 | 12 | 1M | 3mM | |
| NP40 | 40 | 10% | 0.1% | |
| 2M Sucrose | 640 | 2M | 0.32M | |
| RNaseOUT | 20 | 40 U/mL | 0.2 U/mL | |
| Total | 4020 |
Sucrose Diluting Solution (per sample):
| Reagent | Volume [µL] | C_stock | C_final | |
| Nuclease Free Water | 1083.5 | - | - | |
| Tris-HCl pH 7,4 | 11 | 1M | 10mM | |
| NaCl | 2.2 | 5M | 10mM | |
| MgCl2 | 3.3 | 1mM | 3mM | |
| Total | 1100 |
1.2M Sucrose Solution (per sample):
| Reagent | Volume [µL] | C_stock | C_final | |
| 2M Sucrose | 720 | 2M | 1.2M | |
| Sucrose dil. sol. | 480 | - | - | |
| RNaseOUT | 6 | 40 U/mL | 0.2 U/mL | |
| Total | 1206 |
1M Sucrose Solution (per sample):
| Reagent | Volume [µL] | C_stock | C_final | |
| 2M Sucrose | 500 | 2M | 1M | |
| Sucrose dil. sol. | 500 | - | - | |
| RNaseOUT | 5 | 40 U/mL | 0.2 U/mL | |
| Total | 1005 |
Nuclei Wash Buffer (per sample):
| Reagent | Volume [µL] | C_stock | C_final | |
| DPBS -/- | 3593 | - | - | |
| BSA | 257 | 7.5% | 0.5% | |
| RNaseOUT | 19.25 | 40 U/mL | 0.2 U/mL | |
| Total | 3869.25 |
Tissue fixation
15m
1) Add 300 µL of cooled Fixation Solution to brain tissue (1/4 of a mouse brain) in a 1.5mL tube kept on dry ice.
2) Put On ice and start timing.
3) Crush brain(s) with a disposable pellet pestle.
4) Transfer the suspension using a 1mL wide-bore tip into a 2mL tube containing 1.6 mL of cooled Fixation Solution.
5) Shake the sample On ice at 300 rpm . Incubate no longer than00:10:00 (counting from step 2).
6) Add 0.1 mL of 2.5M Glycine (final concentration of glycine is 0.125M).
7) Mix the suspension manually by inverting.
8) Keep the sample On ice and shake at 300 rpm for 00:05:00 .
15m
Nuclei isolation
2h
1) Spin the cell suspension at 1100 x g for 00:05:00 at 2 °C .
2) Remove the supernatant by pipetting.
3) Resuspend the pellet in 1 mL of ice-cold Lysis Buffer using 1mL wide-bore tips (first wash).
4) Spin the cell suspension at 1100 x g for 00:05:00 at 2 °C .
5) Remove the supernatant by pipetting.
6) Resuspend the pellet with 1 mL of ice-cold Lysis Buffer using 1mL wide-bore tips (second wash).
7) Spin the cell suspension at 1100 x g for 00:05:00 at 2 °C .
8) Remove the supernatant by pipetting.
9) Resuspend the pellet with 1 mL of ice-cold Lysis Buffer using 1mL wide-bore tips.
10) Transfer the mixture into a pre-cooled Dounce homogenizer.
11) While keeping homogenizer On ice , homogenize with the Pestle A (5 times for mouse / 15 times for human).
12) While keeping homogenizer On ice , homogenize with the Pestle B - 10 times.
13) Transfer the homogenized solution onto a 30µm filter placed in 2mL LoBind tube.
14) Rinse the homogenizer with 500 µL of the Lysis Buffer and transfer it onto the same filter.
15) Incubate the samples On ice for 00:30:00 .
16) Prepare a 2mL tube with 500 µL of 1.2M Sucrose Solution.
17) Carefully transfer the entire volume of cell suspension onto the 1.2M Sucrose Solution (avoid mixing) (Fig. 1).
Fig. 1 : Layering of the nuclei suspension on top of 1.2M Sucrose Solution.
18) Centrifuge at 5000 x g , Brake and Acceleration (1 , 9) for 00:20:00 at 2 °C .
19) Meanwhile, prepare a 1.5 mL tube with 500 µL of 1.2M Sucrose Solution.
20) After centrifugation, aspirate the upper phase and most of lower phase, leaving 100-200 µL .
21) Resuspend the remaining volume in 1 mL of 1M Sucrose Solution.
22) Carefully transfer the entire mix onto the 1.2M Sucrose Solution prepared in step 19 (avoid mixing).
23) Centrifuge at 5000 x g , Brake and Acceleration (1 , 9) for 00:20:00 at 2 °C .
24) Aspirate upper phase and most of lower phase, leaving 100-200 µL .
25) Resuspend the remaining volume in 1 mL of Nuclei Wash Buffer.
26) Centrifuge at 1100 x g , Brake and Acceleration (9 , 9) for 00:05:00 at 2 °C .
27) Remove the supernatant.
28) Resuspend the pellet in 500 µL of Nuclei Wash Buffer.
29) Incubate the samples On ice for 00:30:00 .
2h
Staining of nuclei
10m
1) Transfer the desired amount of nuclei suspension into a 1.5 mL tube.
2) Add antibodies in recommended ratios
| Ab - marker | Ord No | Fluorochrome | Producer | Dilution | |
| ACSA-2 | 130-116-245 | APC | Milteneyi | 1:160 | |
| NeuN | Mab 377X | AF-488 | Merck | 1:400 | |
| PU.1 | 81886S | PE | CST | 1:400 |
3) Leave some volume of suspension without antibodies as Negative Control.
4) Incubate the suspension with antibodies Overnight at 4 °C , protected from light.
After night:
5) Centrifuge at 1100 x g for 00:05:00 at 2 °C .
6) Remove the supernatant.
7) Resuspend the nuclei in 1 mL of Nuclei Wash Buffer.
8) Centrifuge all samples along with the Negative Control at 1100 x g for 00:05:00 at 2 °C
9) Remove the supernatant.
10) Resuspend nuclei in 1 mL Nuclei Wash Buffer.
11) Filter the suspension through a 30 µm filter.
10m
Sorting and processing according to Smart-seq3xpress protocol
1h 15m
Use FACS to sort nuclei into 384-well plates filled with the modified Lysis Buffer Mix* prepared for Smart-seq3xpress protocol, according to the gating strategy (Fig. 2).
Fig. 2: Gating strategy for sorting
* Smart-seq3xpress protocol has several changes in comparison with the original protocol:
1) Preparation of Lysis Buffer Mix :
a) with RNaseOUT as RNase inhibitor:
| Reagent | Volume [µl] per well | Volume [µl] per 384-well plate | C_stock | C_final | |
| Low TE buffer | 0.215 | 107.5 | - | - | |
| dNTP | 0.02 | 10 | 10mM | 0.5mM | |
| Oligo dTVN30 | 0.005 | 2.5 | 10µM | 0.125µM | |
| SIRV - dilution 1:2000 | 0.001 | 0.5 | - | - | |
| PEG 8000 | 0.05 | 25 | 40% | 5% | |
| Thermolabile proteinase K | 0.006 | 3 | 120U/mL | 2.4U/mL | |
| RNaseOUT | 0.003 | 1.5 | 40U/µL | 0.4U/µL | |
| Total | 0.3 | 150 |
b) with SEQURNA as RNase inhibitor:
| Reagent | Volume [µl] per well | Volume [µl] per 384-well plate | C_stock | C_final | |
| Low TE buffer | 0.2168 | 108.4 | - | - | |
| dNTP | 0.02 | 10 | 10mM | 0.5mM | |
| Oligo dTVN30 | 0.005 | 2.5 | 10µM | 0.125µM | |
| SIRV - dilution 1:2000 | 0.001 | 0.5 | - | - | |
| PEG 8000 | 0.05 | 25 | 40% | 5% | |
| Thermolabile proteinase K | 0.006 | 3 | 120U/mL | 2.4U/mL | |
| SEQURNA | 0.0012 | 0.6 | 50U/µl | 0.2U/µl | |
| Total | 0.3 | 150 |
2) Cell lysis protocol on thermocycler was changed for proteinase digestion and denaturation:
First, incubate the plate for 00:30:00 at 37 °C ,
followed by 00:30:00 at 56 °C ,
then 00:15:00 at 80 °C .
and finally 4 °C hold.
3) The number of cycles during Preamplification PCR is set to 15.
1h 15m