Feb 01, 2026
Version 1
(P75) Isolation of single nuclei from frozen tissues V.1
- Yunming Wu1
- 1HHMI/Stanford University
Protocol Citation: Yunming Wu 2026. (P75) Isolation of single nuclei from frozen tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbo2eylpk/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: In development
We are still developing and optimizing this protocol
Created: January 31, 2026
Last Modified: February 02, 2026
Protocol Integer ID: 242381
Keywords: snRNA-Seq, single cell, RNA-Seq, single nucleus RNA-Seq, single cell RNA-Seq, scRNA-Seq, nuclei isolation, nuclei cryoperservation, preparation of cryopreserved sample, frozen tissues this protocol, isolation of single nuclei, cryopreserved sample, reuse of the same nuclei batch, extracted nuclei, same nuclei batch, nucleus rna, frozen tissue, nuclei for future use, single nuclei, fresh tissue access, snrna
Funders Acknowledgements:
Howard Hughes Medical Institute
Disclaimer
Please note that this protocol is not peer-reviewed and is provided without warranty. Users should validate the method for their specific sample types by assessing RNA integrity and yield. Known issues of this protocol have been annotated as "CRITICAL" notes behind the steps. Not following these critical notes may result in undesirable results.
Abstract
This protocol details the preparation of cryopreserved samples for single-nucleus RNA sequencing (snRNA-seq) and the subsequent storage of extracted nuclei for future use. This workflow is particularly advantageous when fresh tissue access is limited or when longitudinal study designs require the reuse of the same nuclei batches.
Materials
UltraPure, 1M, pH 8.0, Tris-HCl
ThermoFisher, 15568025
Sucrose
Sigma S0389
KCl
Sigma P5405
1M MgCl2
ThermoFisher, AM9530G
10% Triton X-100
Sigma, 648463
RNasin Plus RNase Inhibitor
Promega, N2611
Protease inhibitor
Promega G6521
100 mM DTT
ThermoFisher, 707265
BSA
VWR, 0332
Ultrapure, H2O
ThermoFisher, 10977
Protector Rnase Inhibitor
Roche, 3335399001
10X PBS
ThermoFisher, 70011
Dounce homogenizer
Fisher, 06-434
Anti-NeuN Antibody, clone A60, Alexa Fluor 488 conjugated
Sigma, MAB377X
Kynurenic acid sodium salt
Hellobio, HB0363
NaH2PO4
Sigma, S5011
HEPES
Sigma, H3375
Sodium ascorbate
Sigma, A4034
Thiourea
Sigma, T7875
Sodium pyruvate
Sigma, P5280
CaCl2
Sigma, C5670
Taurine
Sigma, T8691
Myo-inositol
Sigma, I5125
NMDG
Sigma, M2004
HCl
Sigma, 258148
APV
Sigma, A8054
DNQX
Sigma, D0540
TTX
Tocris, 1078
D-(+)-Trehalose dihydrate
Sigma, 90210
D-(+)-Glucose
Sigma, G7021
Sodium Bicarbonate 7.5% solution
ThermoFisher, 25080094
O.C.T.
Fisher Scientific, NC1862249
DMSO
ThermoFisher, D12345
pluriStrainer Mini 40 µm (Cell Strainer)
PluriSelect, 43-10040-60
pluriStrainer Mini 70 µm (Cell Strainer)
PluriSelect, 43-10070-60
Draq5
Biolegend, 424101
SiR-DNA
Cytoskeleton, CY-SC007
Troubleshooting
Before you start
45m
Prepare 50 mL NIM1 by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| 1M Tris pH8.0 | 10 mM | 500 µL | |
| Sucrose | 250 mM | 4.26 g | |
| KCl | 50 mM | 0.1864 g | |
| 1M MgCl2 | 5 mM | 250 µL | |
| H2O | n/a | To 50 mL | |
| Total | n/a | 50 mL |
Note: This solution can be prepared in advance and stored at 4C for 1 month.
5m
Prepare 100 mM taurine by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| Taurine | 100 mM | 0.625g | |
| H2O | n/a | To 50 mL | |
| Total | n/a | 50 mL |
Note: This solution can be prepared in advance and stored at -20C for 1 year.
Prepare 10% BSA by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| BSA | 10% | 1 g | |
| H2O | n/a | To 10 mL | |
| Total | n/a | 10 mL |
Note: This solution can be prepared in advance and stored at -20C for 1 year.
5m
Prepare 100X Kynurenic acid solution by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| Kynurenic acid sodium salt | 100 mM | 1 g | |
| H2O | n/a | To 47.36 mL | |
| Total | n/a | 47.36 mL |
Note: Aliquot into 2mL each, store at -20C.
5m
Prepare 25 mM APV by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| APV | 25 mM | 50 mg | |
| H2O | n/a | To 10 mL | |
| Total | n/a | 10 mL |
Note: Aliquot into 200 µL each, store at -20C.
5m
Prepare 100 mM DNQX by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| DNQX | 100 mM | 50 mg | |
| DMSO | n/a | 1.68 mL | |
| Total | n/a | 1.68 mL |
Note: Aliquot into 200 µL each, store at -20C.
5m
Prepare 100µM TTX by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| TTX | 100 uM | 1 mg | |
| H2O | n/a | 31 mL | |
| Total | n/a | 31 mL |
Note: Aliquot into 200 µL each, store at -20C.
5m
For vertebrates including mice, voles, lizards, frogs, and fish:
Prepare 10X ACSF by following the table below.
| A | B | C | |
| Reagent | Final concentration (mM) | Amount (g) | |
| KCl | 25 | 1.864 | |
| NaH2PO4 | 12 | 1.44 | |
| HEPES | 200 | 47.66 | |
| Sodium ascorbate | 50 | 9.9 | |
| Thiourea | 20 | 1.522 | |
| Sodium pyruvate | 30 | 3.3 | |
| CaCl2 | 10 | 1.11 | |
| 1 M MgCl2 | 13 | 13.17 mL | |
| 100 mM Taurine | 0.1 | 1 mL | |
| Myo inositol | 30 | 5.4 | |
| NMDG | 960 | 187.4 | |
| 35% HCl | n/a | pH to 7.3 | |
| H2O | n/a | To 1 L | |
| Total | n/a | 1 L |
Note: Store the 10X ACSF in 20 mL/tube aliquot at -20C. The thiourea turns yellow when ACSF expires.
For invertebrate, fruit fly:
Prepare 10X AHL by following the table below.
| A | B | C | |
| Reagent | Final concentration (mM) | Amount (g) | |
| KCl | 50 | 3.728 | |
| NaH2PO4 | 10 | 1.2 | |
| HEPES | 50 | 11.9 | |
| Sodium ascorbate | 50 | 9.9 | |
| Thiourea | 20 | 1.522 | |
| Sodium pyruvate | 30 | 3.3 | |
| CaCl2 | 20 | 2.22 | |
| 1M MgCl2 | 82 | 83 mL | |
| 100 mM Taurine | 0.1 | 1 mL | |
| Myo inositol | 30 | 5.4 | |
| NaCl | 1000 | 58.44 | |
| 35% HCl | n/a | pH to 7.5 | |
| H2O | n/a | To 1 L | |
| Total | n/a | 1 L |
Note: Store the 10X AHL in 20 mL/tube aliquot at -20C. The thiourea turns yellow when AHL expires.
15m
Prepare solution.
22m
For vertebrates including mice, voles, lizards, frogs, and fish:
Prepare 1X ACSF by following the table below in order.
| A | B | C | |
| Reagent | Final concentration (mM) | Amount (g) | |
| H2O | n/a | 150 mL | |
| 10X ACSF | 1X | 20 mL | |
| Glucose | 20 | 0.9 g | |
| Trehalose | 73 | 5 g | |
| 25 mM APV | 0.025 | 200 µL | |
| 100 µM TTX | 0.1 | 200 µL | |
| 100 mM DNQX | 0.1 | 200 µL | |
| 100 mM Kynurenic acid | 1 | 2 mL | |
| H2O | n/a | to 200 mL | |
| Total | n/a | 200mL |
CRITICAL: This solution needs to be prepared fresh. The pH should be arround 7.3. Osmolarity should be arround 300-310 mOsm.
For invertebrates, fruit fly:
Prepare 1X AHL by following the table below in order.
| A | B | C | |
| Reagent | Final concentration (mM) | Amount (g) | |
| H2O | n/a | 150 mL | |
| 10X ACSF | 1X | 20 mL | |
| Sucrose | 10 | 0.685 g | |
| Trehalose | 5 | 0.378 g | |
| 25 mM APV | 0.025 | 200 µL | |
| 100 µM TTX | 0.1 | 200 µL | |
| 100 mM DNQX | 0.1 | 200 µL | |
| 100 mM Kynurenic acid | 1 | 2 mL | |
| H2O | n/a | to 200 mL | |
| Total | n/a | 200mL |
CRITICAL: This solution needs to be prepared fresh. The pH should be arround 7.5. Osmolarity should be arround 260-280 mOsm.
10m
Place the solution on ice.
1m
Oxygenate the solution with carbogen (95% O₂, 5% CO₂) for 5 minutes prior to the next step, maintaining continuous bubbling thereafter.
5m
For vertebrates including mice, voles, lizards, frogs, and fish:
Add 6mL 7.5% NaHCO3.
For invertebrates, fruit fly:
Add 896 µL 7.5% NaHCO3.
1m
Add ddH2O to 200mL.
Note: the prepared solution is kept on ice with continuous oxygenation through the entire experiment.
5m
Tissue dissection
33m
Sacrifice the animal using CO2 and cervical dislocation.
15m
Dissect the brain in ice cold ACSF/AHL prepared in step 12.
Embed into O.C.T..
5m
Float a PCR rack on liquid nitrogen.
1m
Place the O.C.T. block on the PCR rack.
Critical: Placing the O.C.T. block directly into the liquid nitrogen will break the block.
1m
Wait until the O.C.T. block completely freezes.
10m
Store the snap frozen tissue under -80C.
1m
Nuclei extraction
2h 38m
Prepare NIM2 by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| NIM1 | 1 X | 4.9 mL | |
| 100 mM DTT | 0.1 mM | 5 µL | |
| 50X protease inhibitor | 1 X | 100 µL | |
| 10% BSA | 0.1% | 50 µL | |
| Total | n/a | 5 mL |
2m
Prepare NIM3 by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| NIM2 | 1 X | 1 mL | |
| Protector RNAse inhibitor/or RNasin plus | 0.2 unit/µL | 5 µL | |
| Total | n/a | 1 mL |
Note: 1 mL of NIM3 is used per sample.
2m
Prepare nuclei freezing medium by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| NIM2 | 90% | 900 µL | |
| Protector RNAse inhibitor/or RNasin plus | 0.2 unit/µL | 5 µL | |
| DMSO | 10% | 100 µL | |
| Total | n/a | 1 mL |
2m
Place the Dounce homogenizer (Dounce for short from here on) on ice.
1m
Add 1mL NIM3 to a Dounce.
1m
Cryosection the O.C.T. block into 200 um sections.
1h
Keep the section cold and place the section onto dry ice plate.
1m
Dissect the region out while the section is frozen under a microscope.
1h
Transfer the dissected tissue into the Dounce on ice.
Critical: the amount of tissue should be limited. Too much tissue at this step will result in failed extraction. A general starting point is 50mg in weight or 2mm in size.
2m
Dounce on ice with loose pestle 10 times,
1m
Dounce on ice with tight pestle 5 times.
CRITICAL: For tissue type used the first time, the number of Dounce should be determined by a few trial experiment. Optimal number is determined by generating the largest amount of nuclei while maintaining the integrity of the nuclei morphology.
1m
Centrifuge 400g, 4C, 5min.
5m
Remove supernatant.
1m
Add 100 µL of 10% BSA into 900 µL of NIM3 to make 1%BSA cushion.
1m
Transfer the 1 mL 1%BSA cushion into a 5mL polypropylene flow tube.
1m
Add 10 µL 10% Triton X-100 to 1mL NIM3, mix gently to avoid bubbles.
1m
Use the NIM3 with tritonx100 to slowly resuspend pellet by pipetting up and down 3 times.
1m
On ice 1 minute.
1m
Filter through 70 µm filter.
1m
Filter through 40 µm filter.
1m
Carefully lay the cell lysate on the top of the cushion.
1m
Centrifuge 400g, 4C, 5min.
5m
Resuspend the pellet into 1mL nuclei freezing medium.
Optional: check the nuclei quality and quantity here. Good quality nuclei should look round, with minimal visible tissue debris, without small membranes, and abundant in quantity. Most if not all of the nuclei should be stained with cell nonpermeable dyes.
1m
Aliquot into 10-20 tubes depending on the need.
5m
Store the aliquots at -80C.
Nuclei staining, selection of neuronal nuclei, and FANS purification
2h 5m
Prepare nuclei staining buffer by following the table below.
| A | B | C | |
| Reagent | Final concentration | Amount | |
| 10X PBS | 1X | 250 uL | |
| 10% BSA | 1% | 250 uL | |
| ddH2O | n/a | 2000 uL | |
| Protector RNAse inhibitor/or RNasin plus | 0.2 unit/uL | 12.5 uL | |
| Total | n/a | 2.5 mL |
5m
Thaw the DMSO stored samples on ice.
10m
Add 1000 µL staining buffer and gently mix by slowly pipetting up and down.
1m
Centrifuge 400g, 4C, 5min.
5m
Discard supernatant.
1m
Resuspend in 200 µL staining buffer.
1m
Add 1 µL NeuN antibody.
1m
4C incubate 20 minutes.
20m
Add 800 µL staining buffer.
1m
Centrifuge 400g, 4C, 5min.
5m
Discard supernatant.
1m
Resuspend in 500 µL staining buffer
1m
Add 0.5 µL 1000X SiR-DNA or Draq5.
1m
Filter through 40 µm filter.
1m
Briefly examine quality by mixing with propidium iodide and trypan blue.
10m
Wet a 1.5 ml tube with 18.8 µI RT reagent B.
1m
Sort 24000 cells using 70µm nozzle and lowest sample pressure (=43.3 µl) into 18.8 RT mix. For bigger nuclei (e.g. frog), 18000 nuclei using 100µm nozzle (= 43.3 µl) into 18.8 RT mix.
CRITICAL: One should try to use the lowest possible sorting pressure possible to avoid breaking the nuclei during the sorting.
1h
Proceed to 10X droplet generation.