Mar 18, 2026
Nuclei Isolation Protocol from Fresh and Frozen Tissue for Single-Nucleus RNA-Seq and ATAC-Seq
- Zia Hasan1,
- Touati Benoukraf1,
- Qi Yuan1
- 1Memorial University of Newfoundland
- scEpiTrans
Protocol Citation: Zia Hasan, Touati Benoukraf, Qi Yuan 2026. Nuclei Isolation Protocol from Fresh and Frozen Tissue for Single-Nucleus RNA-Seq and ATAC-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn228zg5d/v1
Manuscript citation:
Hasan, Z., Torraville, S. E., Omoluabi, T., Maziar, A., Belolise, O. N., MacGowan, L. A., ... & Yuan, Q. (2026). Sex and life experience shape locus coeruleus pretangle tau pathology. Alzheimer's & Dementia, 22(3), e71285.
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: October 30, 2022
Last Modified: March 18, 2026
Protocol Integer ID: 72051
Keywords: nuclei isolation, fresh tissue, frozen tissue, snRNA-seq, single-nucleus ATAC-seq, snATAC-seq, nuclear extraction, tissue dissociation, nuclei quantification, hippocampus, brain tissue, single cell rna seq, single nucleus RNA seq, single nucleus ATAC seq, nuclei isolation protocol from fresh, established nuclei isolation method, nuclei isolation method, nuclei isolation protocol, isolating nuclei, nuclei recovery, assessment of nuclei quality, quality nuclei suspension, preserving nuclear membrane integrity, nuclei quality, nucleus rna, nuclear membrane integrity, cost manual nuclei, manual nuclei, frozen tissue for single, rna degradation, nucleus application, fluorescence microscope, frozen tissue
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Abstract
This protocol describes an optimized method for isolating nuclei from fresh and frozen tissue for single-nucleus RNA-seq and ATAC-seq. The workflow integrates key steps from several established nuclei isolation methods and includes modifications to improve nuclei recovery, reduce RNA degradation, and minimize debris and contamination. Optimization focused on tissue-to-buffer ratio and lysis duration to achieve efficient dissociation while preserving nuclear membrane integrity. The protocol also includes a simple, low-cost manual nuclei counting method using a fluorescence microscope, allowing assessment of nuclei quality and concentration without the need for an automated counter. This workflow is intended to provide a practical and accessible approach for generating high-quality nuclei suspensions for downstream single-nucleus applications.
Materials
Buffer and Reagents:
8. Ethidium Bromide/ethidium homodimer-1/DAPI solution
Stock Buffers:
Add an appropriate amount of reagents in Nuclease-Free water to get the stock concentration.
| A | B | C | D | |
| Reagent | Amount | Nuclease-Free Water | Stock Conc. | |
| Tricine | 8.96 g | 100 ml | 500 mM | |
| Kcl | 7.45 g | 100 ml | 1M | |
| MgCl2.6H2O | 20.3 g | 100 ml | 1M | |
| KOH | 5.61 g | 100 ml | 1M |
Debris Removal Buffer 1 (DRB1): Keep at Room temperature, Shelf life 2 months
| A | B | C | |
| Reagent | Stock Conc. | Amount | |
| Nuclease-Free Water | - | 50 ml | |
| Tricine | 500mM | 24 ml | |
| Kcl | 1M | 15 ml | |
| MgCl2.6H2O | 1M | 3 ml | |
| Adjust the pH to 7.8 with 1M KOH | 1M | - | |
| Add Nuclease-Free Water to make up to 100 ml | - | - | |
| Total | 100 ml |
Debris Removal Buffer 2 (DRB2): Keep at 4°C, Shelf life 2 months)
| A | B | C | |
| Reagent | Stock Conc. | Amount | |
| Nuclease-Free Water | - | 50 ml | |
| Glucose | 8.5 gm | ||
| Dissolve the Glucose in water then add the following | |||
| Tricine | 500mM | 4 ml | |
| Kcl | 1M | 2.5 ml | |
| MgCl2.6H2O | 1M | 0.5 ml | |
| Adjust the pH to 7.8 with 1M KOH | 1M | - | |
| Add Nuclease-Free Water to make up to 100 ml | - | - | |
| Total | 100 ml |
Working Buffers
Debris Removal Working Buffer (DRWB): (Prepare Fresh on Ice)
| A | B | C | |
| Reagent | For 1 sample | For 4 samples | |
| OptiPrep™ - Iodixanol (Iodixanol: 60% (w/v) in water) | 1.25 | 5 | |
| DRB1 | .25 | 1 | |
| Vortex for a few seconds | |||
| DRB2 | 1 | 4 | |
| Vortex for a few seconds | |||
| Total | 2.5 ml | 10 ml |
Lysis Buffer: (Prepare Fresh on Ice)
| A | B | C | |
| Reagent | stock Conc. | Working Conc. | |
| Nuclei EZ Prep | - | - | |
| RNase Inhibitor | 40 U/uL | 0.2 - 1U/μL | |
| DTT | 1 M | 1 mM |
Example: For preparing Wash and Resuspension Buffer with 0.3 U/μL RNAse Inhibitor for 4 samples, I prepare 10 ml of Lysis Buffer (Prepare Fresh and keep on Ice).
| A | B | C | D | |
| Reagent | stock Conc. | Amount | Working Conc. | |
| Nuclei EZ Prep | - | 9925 uL | - | |
| RNase Inhibitor | 40 U/uL | 75 uL | 0.3 U/μL | |
| DTT | 1 M | 10 uL | 1 mM | |
| Total | 10,000 uL |
Wash and Resuspension Buffer: (Prepare Fresh on Ice)
| A | B | C | |
| Reagent | stock Conc. | Working Conc. | |
| PBS | 1X | 1X | |
| MACS® BSA Stock Solution | 10% | 1% | |
| RNase Inhibitor | 40 U/uL | 0.2- 1 U/uL |
Example: For preparing Wash and Resuspension Buffer with 0.4 U/μL RNAse Inhibitor for 4 samples, I prepare 13 ml of Wash and Resuspension Buffer (Prepare Fresh and keep on Ice).
| A | B | C | D | |
| Reagent | stock Conc. | Amount | Working Conc. | |
| PBS | 1X | 11,570 uL | 1X | |
| MACS® BSA Stock Solution | 10% | 1300 uL | 1% | |
| RNase Inhibitor | 40 U/uL | 130 μL | 0.4 U/μL | |
| Total | 13,000 uL |
Nuclei Quality Check and count medium:
| A | B | |
| Reagent | 1X | |
| Trypan Blue Solution, 0.4% | 11.4 μL | |
| Ethidium Bromide/ethidium homodimer-1/DAPI solution | 0.6 μL | |
| Total | 12 μL |
Example: For preparing Nuclei Quality Check and count medium for 4 samples, I prepare 60 μL of Nuclei Quality Check and count medium.
| A | B | |
| Reagent | 5X | |
| Trypan Blue Solution, 0.4% | 57.0 μL | |
| Ethidium Bromide/ethidium homodimer-1/DAPI solution | 3.0 μL | |
| Total (vortex and centrifuge for a few seconds) | 60 μL |
Troubleshooting
Tissue Dissociation and Nuclei Isolation
15m
Using a sterile single-edge blade or a surgical scalpel with a handle, carefully fragment the tissue into small pieces roughly the size of rice grains. Use tweezers to hold the tissue in place and prevent it from scattering. Once fragmented, transfer the tissue into a 1.5 mL, 2 mL, or 5 mL microcentrifuge tube.
Note
Do not thaw the tissue before or during dissociation to prevent RNA degradation. Instead, place the Petri dish or glass surface on dry ice and mince the tissue while it remains frozen.
• tissue weight < 50 mg, use a 1.5 mL microcentrifuge tube.
• tissue weight > 50 mg and up to 100 mg, use a 2- or 5-mL microcentrifuge tube.
Add 300-500 µL of chilled Nuclei EZ Lysis Buffer, supplemented with RNAse Inhibitor (0.2–1 U/µL) and DTT (1 mM), to the minced tissue in a 1.5 mL or 2 mL microcentrifuge tube.
Note
• tissue weight < 50 mg, add 300 µL of Lysis Buffer.
• tissue weight > 50–100 mg, add 500 µL of Lysis Buffer.
• tissue weight > 100 mg, adjust the Lysis Buffer volume accordingly.
Homogenize the tissue using a pre-chilled plastic pestle by gently pressing it down and twisting or lifting it against the inner wall of the tube. Ensure the tube always remains on ice during this process. If needed, briefly remove the tube from the ice to monitor tissue dissociation, but promptly return it to the ice to maintain optimal conditions. After homogenization, incubate the tube on ice00:05:00 (1st incubation).
5m
Add 700-1500 µL of chilled Nuclei EZ Lysis Buffer to the tube. Pipette mix 5x with 1mL wide-bore pipette tips (wide-bore tips can be made by cutting the ends of standard 1 mL tips). If the pipette tip becomes clogged with unhomogenized tissue, continue to dissociate tissue with the pestle until able to pipette mix.
Note
• tissue weight < 50 mg, add 700 µL of Lysis Buffer.
• tissue weight > 50 mg and up to 100 mg, add 1000–1500 µL of Lysis Buffer.
• tissue weight > 100 mg, adjust the Lysis Buffer volume accordingly.
Incubate on ice for 00:05:00 ; repeat mixing 2-3 times (5x) during the incubation (use standard 1ml tips).
Note
The 2nd incubation time depends on the tissue type. While most tissues or cells typically lyse within00:05:00 , optimization may be necessary to determine the optimal lysis time for specific tissue types. In our experiments:
• The olfactory bulb required an incubation time of 00:10:00
• The hippocampus required an incubation time of 00:15:00
To identify the appropriate lysis time for a tissue type, perform a lysis timeline by testing different incubation durations to ensure complete and efficient tissue dissociation.
Optimization of tissue lysis time (Hippocampus). To determine the
optimal lysis duration, we performed a time-course experiment (10, 15, and 20
mins). We found that 15 mins provided the best results for hippocampal tissue,
yielding intact nuclei with minimal debris.
5m
Filter the dissociated tissue using a 70 μm-strainer mesh to fit a 15 ml/50 ml Falcon tube (e.g., pluriStrainer Mini 70 μm Cell Strainer).
If a swinging bucket centrifuge is available, use the 15ml/50ml test tube. Or transfer flow through into a 1.5 mL- 2 ml LoBind tube and centrifuge the nuclei at500 x g, 4°C using a standard centrifuge. Carefully remove the supernatant, leaving approximately ~50 µL of the remaining sample.
5m
Debris removal
Carefully remove supernatant as much as possible without disturbing the pellet. Leave ~ 50–100 µL of liquid if the pellet appears loose. Add 300–500 µL of DRWB (vortex a few seconds before adding) and mix gently to resuspend the pellet without creating bubbles.
Take 1 mL of DRWB into a new 1.5 mL tube. Gently layer the crude nuclei + DRWB suspension on top of the DRWB layer by angling the tube slightly and slowly dispensing the suspension along the side wall using a pipette tip. A clear separation between the crude nuclei suspension and the DRWB layer should become visible.
Centrifuge at 10000 x g, 4°C, 00:20:00 . After centrifugation, the debris will float to the top, while the clean nuclei pellet will settle at the bottom or on the sidewall of the tube.
Note
To avoid the nuclei sticking to the side walls of the tube, consider using a swinging bucket centrifuge. Alternatively, rinse 1.5 mL LoBind tubes with 5% BSA solution and allow them to dry before starting the experiment.
In this protocol, a standard centrifuge was initially used, followed by a swinging bucket centrifuge at a maximum speed of 4.500 rpm, 4°C, 00:20:00
20m
Nuclei Wash, Resuspension and Aggregate Removal
10m
Carefully remove the supernatant containing debris using a wide-bore 1 mL pipette tip without disturbing the pellet. Remove as much supernatant as possible, and for any remaining liquid, use a 200 μL pipette for precise removal. Add 1 mL of ice-cold Nuclei Wash and Resuspension Buffer (supplemented with 0.2–0.5 U/μL RNase Inhibitor) to the pellet. Avoid mixing the solution by pipetting. Centrifuge the nuclei at 500 x g, 4°C, 00:05:00
Note
If there are no visible nuclei pellet forms (after step 10), leave approximately ~200 μL of supernatant behind, add 1 mL of ice-cold Nuclei Wash and Resuspension Buffer, and proceed.
5m
If the nuclei pellet is visible, carefully remove as much supernatant as possible without disturbing the pellet. If no pellet is visible, leave ~200 μL of supernatant to avoid losing nuclei. Add 1 mL of ice-cold Nuclei Wash and Resuspension Buffer (supplemented with 0.2–0.5 U/μL RNase Inhibitor) to the pellet and gently pipette mix. Centrifuge the nuclei at 500 x g, 4°C, 00:05:00
5m
Carefully remove the supernatant and resuspend the pellet in 50–500 µL (Depending on the desired concentration) of ice-cold Nuclei Wash and Resuspension Buffer supplemented with 0.2–0.5 U/μL RNAse Inhibitor. Filter the suspension using a 40-µm Flowmi‱ Cell Strainer (300 µL per strainer) and transfer the filtered nuclei suspension to a new LoBind 1.5 mL Eppendorf tube.
Nuclei Qulity check and Counting
An automatic fluorescent cell counter or a fluorescence microscope with a hemocytometer can be used to assess nuclei quality and count. We mixed Trypan blue with Ethidium bromide/DAPI solution to assess the nuclei quality and count simultaneously.
- Prepare the Nuclei Quality Check and Count Medium.
- Take 12 μL of the Nuclei Quality Check and Count Medium and mix it with 12 μL of the nuclei suspension.
Note
Prior to mixing the nuclei suspension, vortex at maximum speed for 2–3 seconds, then immediately pulse-spin for no longer than 1 second to collect the suspension at the bottom of the tube.
- Pipette the mixture gently 5–10 times to ensure proper mixing.
- Load 10 μL of the prepared mixture into each chamber of a C-Chip Disposable Hemocytometer (4 chambers per slide). For each sample, count nuclei in at least two chambers to ensure accuracy.
- Wait for 5–10 seconds to allow the nuclei to settle.
- Examine the sample under a fluorescence microscope (e.g., EVOS M5000 Imaging System).
- Use the RFP (Red) channel to detect nuclei stained with Ethidium Bromide or Ethidium Homodimer-1.
- Use the Blue channel to detect nuclei stained with DAPI.
Nuclear RNA Integrity check
The RNA was extracted from the nuclei suspension to validate the protocol's establishment. The RNA integrity was evaluated by agarose gel electrophoresis and on an Agilent TapeStation, and RNA purity was measured with a NanoDrop spectrophotometer.