Mar 05, 2026
Nuclei Isolation from Rat and Bovine White Adipose Tissue for snRNAseq on Parse Platform
- Janice Thompson1,
- Miguel Chirivi2,
- Leah Terrian3,
- Andres Contreras2,
- Stephanie Watts1,
- Rance Nault1
- 1Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan, United States;
- 2Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, Michigan, United States;
- 3Department of Biomedical Engineering, Michigan State University, East Lansing, Michigan, United States
Protocol Citation: Janice Thompson, Miguel Chirivi, Leah Terrian, Andres Contreras, Stephanie Watts, Rance Nault 2026. Nuclei Isolation from Rat and Bovine White Adipose Tissue for snRNAseq on Parse Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx42kwl8j/v1
Manuscript citation:
A manuscript has been submitted for peer review and will be added when the information becomes available.
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 to isolate sufficient numbers of nuclei from bovine and rat WAT for snRNAseq.
Created: October 08, 2025
Last Modified: March 05, 2026
Protocol Integer ID: 229256
Keywords: white adipose tissue, snRNAseq, nuclei isolation, rat, cattle, performed single nucleus rna, nuclei isolation from rat, bovine white adipose tissue, nuclei isolation protocol for use, nuclei isolation of human wat, nuclei isolation protocol, single nucleus rna, rat mesenteric perivascular adipose tissue, subscapular brown adipose tissue, nucleus rna, white adipose tissue, robust nuclei isolation protocol, same nuclei isolation method, application of the same nuclei isolation method, nuclei isolation, nuclei transcriptomic profile, nuclei protocol, bovine white fat, rat retroperitoneal fat, rna, mesenteric perivascular adipose tissue, quality of nuclei, quantity of nuclei, nuclei, brown fat, useful for snrnaseq, insufficient yield of nuclei, frozen tissue dissociation, same rat strain, white fat, transcriptomic profile, improved lipid removal technique
Funders Acknowledgements:
National Institutes of Health Grants
Grant ID: P01HL152951
Michigan Alliance for Animal Agriculture
Grant ID: Award AA-24-034
Abstract
We had previously successfully performed single nucleus RNA-sequencing (snRNAseq) on brown fats: the thoracic aortic PVAT and subscapular brown adipose tissue (BAT) from Dahl SS rats. However, application of the same nuclei isolation method to white adipose tissue (WAT) depots (perivascular and non-perivascular) from the same rat strain resulted in insufficient yield of nuclei (< 200 nuclei/mg) and RNA for downstream single-nuclei protocols. These challenges were also encountered when processing WAT from cattle, which exhibits difficulties similar to those observed with human WAT. This study aimed to validate and optimize a nuclei isolation protocol for use with both rat and bovine WAT samples. This is important because it is WAT that surrounds the vessels that govern total peripheral resistance and is most associated with an increase in cardiovascular risk. Protocols were evaluated based on a) the quantity of nuclei isolated and b) quality of nuclei determined by microscopic visualization. A protocol utilizing liquid nitrogen pulverization and Dounce homogenization for flash-frozen tissue dissociation and homogenization and wash buffers designed for nuclei isolation of human WAT was tested. This protocol, with key modifications for optimization, proved translatable to rat and bovine WAT depots (rat retroperitoneal fat: 3100 nuclei/mg tissue; rat mesenteric perivascular adipose tissue: 2200 nuclei/mg tissue; bovine white fat: 1450 nuclei/mg tissue). These protocol modifications included improved lipid removal techniques and optimization of centrifugation speed. Collectively, we illustrate the importance of a robust nuclei isolation protocol that is generalizable across distinct adipose depot locations and species for the evaluation of single-nuclei transcriptomic profiles. This developed protocol should prove useful for snRNAseq of WAT from multiple species.
Guidelines
Tissue collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee. This procedure complied with National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011) and was approved by the Michigan State University Institutional Animal Care and use Committee (PROTO202000009).
Materials
Reagents:
- dithiothreitol (DTT; GoldBio, cat. no. DTT10)
- sucrose (Sigma, S-7903)
- 10% Bovine Serum Albumin (10% BSA; Millipore-Sigma, cat. no. A1595-50mL)
- Molecular Grade Nuclease-Free Water (Invitrogen, cat. no. AM9930)
- Triton X-100 (Sigma, cat. no. T8787)
- magnesium chloride (MgCl2; Sigma, cat. no. M8266)
- trizma base (Sigma, cat. no. T1503)
- potassium chloride (KCl; Avantor, cat. no. 3040-05)
- ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA; J.T. Baker, cat. no. 8993-01)
- Halt protease inhibitor 100X (ThermoScientific, cat. no. 87785)
- Superasin (20 U/μL) (Invitrogen, cat. no. AM2696)
- RiboLock RNase Inhibitor (ThermoScientific, cat. no. EO0382)
- DNase (Qiagen, cat. no. 79254)
- liquid nitrogen
- ice
- Phosphate-Buffered Saline without calcium and magnesium (PBS; Millipore-Sigma, cat. no. D8537-500ML)
- acridine orange/propidium iodide stain (AO/PI; Nexcelom, cat. no. CS2-0106-5mL)
2.2 Consumables:
- 2 mL LoBind microcentrifuge tubes (Eppendorf, cat. no. 022431048)
- Mortar and pestle (1 set per sample)
- spatula (1 per sample)
- Dounce homogenizer (1 set per sample)
- 1.5 mL LoBind microcentrifuge tubes (Eppendorf, cat. no. 022431021)
- 20 μL, 200 μL, 1000μL low binding pipet tips (Rainin, cat. no. 30389226, 30389240, 30389213)
- 100 μm filters (VWR, cat. no. 76327-102)
- 40 μm filters (VWR, cat. no. 76327-098)
- 15 mL conical tubes (Corning, cat. no. 430052)
- 50 mL conical tubes (Corning, cat. no. 430290)
- gel loading tips (Fisherbrand, cat. no. 02-707-181)
- 1 mL syringe with 25-gauge needle (Becton Dickinson, cat. no. 309626)
2.3 Equipment
- vortex
- refrigerated centrifuge with 1.7 mL tube capacity
- hemacytometer
- microscope for Brightfield and fluorescence
Troubleshooting
Tissue Collection
Note
The Materials section contains key resources used in Tissue Collection.
Tissues were dissected and placed in beakers containing physiological salt solution [in mM: NaCl 130; KCl 4.7; KH2PO4 1.18; MgSO4*7H2O 1.17; NaHCO3 14.8; dextrose 5.5; CaNa2EDTA 0.03; CaCl2 1.6 (pH 7.2)]: thoracic aorta, subscapular (brown) fat, mesentery, retroperitoneal fat. Following dissection, tissues were placed in a Silastic-coated dish containing PSS and cleaned of blood. For subscapular (brown) fat, care was taken to remove as much adjacent white fat as possible. Thoracic aorta PVAT was removed from the aorta and mesenteric PVAT was removed from the resistance artery/vein pair. Tissues were snap frozen in liquid nitrogen in 1.5 mL microcentrifuge tubes and stored at -80 °C until nuclei isolation was performed.
Reagent Preparation
Prepare reagents.
Note
Stock reagents can be prepared ahead of time and stored under the appropriate conditions for each individual solution.
The Materials section contains key resources used in Reagent Preparation.
1 mM dithiothreitol (DTT): Add 1 mg DTT to 2.82 mL nuclease free water and dissolve. Aliquot 20 µL to 0.5 mL tubes and store at -20 °C .
1.5 M sucrose: Add 20.54 g sucrose to 40 mL nuclease free water and dissolve. Aliquot 1 mL to 1.5 mL tubes and store at -20 °C .
1% BSA nuclease free water: Add 5 mL 10% BSA to 45 mL nuclease free water. Filter through 0.2 µm syringe filter and store at 4 °C .
10% Triton X-100: Add 100 µL 100% Triton X-100 to 900 µL nuclease free water and dissolve. Store at room temperature.
1 M magnesium chloride (MgCl2): Add 9.5211 g MgCl2 to 90 mL nuclease free water and dissolve. Bring final volume to 100 mL and store at room temperature.
1 M Tris Buffer, pH 8.0: Add 12.114 g Trizma base to 90 mL nuclease free water and dissolve. Adjust pH to 8.0. Bring final volume to 100 mL and store at room temperature.
2 M potassium chloride (KCl): Add 14.91 g KCl to 80 mL nuclease free water and dissolve. Bring final volume to 100 mL and store at room temperature.
0.5 M ethylenediaminetetraacetic acid (EDTA): Add 1.46 g EDTA to 8 mL nuclease free water. Bring final volume to 10 mL and store at room temperature.
Prepare Homogenization and Nuclei Resuspension Buffers
Note
Prepare Homogenization Buffer (Table 1) and Nuclei Resuspension Buffer (Table 2) fresh daily from the reagents prepared in "Reagent Preparation" section. Keep both buffers on ice and protected from light to ensure enzyme integrity.
Prepare Homogenization Buffer (Table 1) – KEEP ON ICE AND PROTECT FROM LIGHT
The following is the amount needed for ONE sample. Space is provided for calculation of specific numbers of samples and volumes.
| Reagent | Volume (µL) | # of Samples | Total Volume (µL) | Final Concentration | |
| 1 M MgCl2 | 15 | 5 mM | |||
| 1 M Tris Buffer, pH 8.0 | 30 | 10 mM | |||
| 2 M KCl | 37.5 | 25 mM | |||
| 1.5 M Sucrose | 500 | 250 mM | |||
| 1 mM DTT | 3 | 1 µM | |||
| Protease Inhibitor (100X) | 30 | 1X | |||
| Superasin (20 U/µL) | 30 | 0.2 U/µL | |||
| nuclease-free water | 2354.5 |
Table 1. Homogenization Buffer
Total Volume: 3000 µL per sample
Prepare Nuclei Resuspension Buffer (Table 3 – needs to be moved to table section at end) – KEEP ON ICE AND PROTECT FROM LIGHT
The following is the amount needed for ONE sample. Space is provided for calculation of specific numbers of samples and volumes.
| Reagent | Volume (µL) | Sample # | Total Volume (µL) | Final Concentration | |
| 1 M MgCl2 | 10 | 5 mM | |||
| 1 M Tris Buffer, pH 8.0 | 20 | 10 mM | |||
| 2 M KCl | 25 | 25 mM | |||
| 0.5 M EDTA | 4 | 1 mM | |||
| Ribolock RNAse Inhibitor | 20 | 0.2 U/µL | |||
| DNase I | 14.7 | 40 units | |||
| 1% BSA nuclease free water | 1906.3 |
Table 2. Nuclei Resuspension Buffer
Total Volume: 2000 µL per sample isolated
Tissue Pulverization and Homogenization
35m 2s
Note
Before beginning, obtain buffer reagents as indicated in “Reagent Preparation”. Prepare Homogenization Buffer and Nuclei Resuspension Buffer fresh daily. Keep on ice and protected from light. Obtain liquid nitrogen and frozen tissue samples.
One sample at a time was pulverized and homogenized, with a maximum of four samples processed at one time to avoid sample degradation. Work from * to * before beginning the next sample.
Safety information
When working with liquid nitrogen, wear appropriate personal protective equipment (PPE) as designated by your institution and local/state/federal guidelines.
Ensure care is taken to keep all tissue pieces inside the mortar when working with liquid nitrogen.
*Cool mortar, pestle, and spatula with liquid nitrogen. Place the pestle in the mortar to ensure adequate cooling of both pieces.
Allow liquid nitrogen to fully evaporate, then add another volume to the mortar.
While cooling, weigh an appropriate amount of tissue: approximately 150 mg for rat and approximately 350 mg for cow. Record values for future normalization.
Add weighed tissue to the liquid nitrogen in the mortar and tap gently with the pestle to break into smaller pieces as the liquid nitrogen evaporates.
Following liquid nitrogen evaporation, pulverize the tissue thoroughly with the pestle, taking care to keep all tissue pieces in the mortar.
Note
Tissue should be a fine frozen powder before proceeding.
Using a spatula, scoop the tissue into a glass Dounce homogenizer.
Add 2 mL of Homogenization Buffer to the tissue and homogenize 10 strokes.
Equally distribute the homogenate between (2) 1.7 mL protein LoBind tubes and place on ice.
Note
LoBind microcentrifuge tubes from Eppendorf have been verified with this protocol. Comparable tubes have not and should be evaluated by the user prior to incorporation into the procedure.
Add 400 µL of Homogenization Buffer to the Dounce homogenizer and pass three strokes to retrieve residual homogenate from both pieces.
Equally distribute the remaining homogenate between the same (2) 1.7 ml LoBind tubes with the samples.*
Repeat from * to * until all samples are homogenized, distributed between 2 LoBind tubes, and on ice.
Add 12 µL 10% Triton X-100 to each 1.7 mL sample tube and pulse vortex tubes for 00:00:02 .
2s
Incubate samples on ice for 00:30:00 , covered from light, with pulse vortexing every 00:05:00 .
35m
While samples are incubating, prepare filters:
For each cow sample isolated, place a 100 µm, 40 µm filter, and 20 µm filter into separate 50 mL conical tubes and pre-wet each filter with 100 µL phosphate buffered saline (PBS).
For each rat sample isolated, place a 100 µm and 40 µm filter into separate 50 mL conical tubes and pre-wet each filter with 100 µL phosphate buffered saline (PBS).
Sample Cleanup
30m
When incubation is complete, filter samples:
Pipette homogenate through 100 μm filter into 50 mL conical tube.
Add 400 µL Homogenization Buffer to the 1.7 mL tube to wash it.
Pipette the wash through the 100 μm filter.
Transfer homogenate from 50 mL conical tube through the 40 μm filter into the 50 mL conical tube.
For rat samples, transfer homogenate equally to (2) 1.7 mL LoBind tubes.
For cow samples, transfer homogenate from 50 mL conical tube through the 20 µm filter into the 50 ml conical tube. Transfer homogenate equally to (2) 1.7 mL LoBind tubes.
Centrifuge the homogenates at 2700 x g for 00:10:00 at 4 °C .
10m
Pipette off and discard lipid layer using a gel loading tip.
Note
The gel loading tip provides precision for lipid removal.
Pipette off and discard remaining supernatant, leaving ~ 50 µL .
Place tube with pellet in it on ice and repeat lipid and supernatant removal with remaining tube(s).
Combine homogenates for each sample from two tubes to one:
Resuspend first pellet in 50 µL supernatant by pipetting up and down 30 times.
Transfer homogenate to new 1.7 mL LoBind tube@.
Resuspend second pellet in 50 µL supernatant by pipetting up and down 30 times.
Transfer to LoBind tube@.
Repeat for all remaining samples.
Add 100 µL Homogenization Buffer to homogenate and mix by pipetting 5 times.
Centrifuge at 2700 x g for 00:10:00 at 4 °C .
Note
There should be a visible pellet and no lipid at the top. If lipid remains, use a gel loading tip to remove and discard.
10m
Remove and discard supernatant, leaving ~ 50 µL .
Resuspend pellet in 50 µL supernatant by pipetting up and down 30 times.
Add 950 µL Nuclei Resuspension Buffer and mix by pipetting 10 times.
Centrifuge at 2700 x g for 00:10:00 at 4 °C .
Note
Following nuclei counting, if too many nuclei appear damaged, this centrifugation step can be reduced to 1000 x g.
10m
Remove and discard supernatant, leaving ~50 µL .
Resuspend pellet in 50 µL supernatant by pipetting up and down 30 times.
Add 450 µL Nuclei Resuspension Buffer and mix by pipetting 10 times.
Using a 1 mL syringe with a 25 gauge needle, pull nuclei solution up and down 10 times to reduce clumping.
Place samples on On ice while staining for nuclei count/integrity.
Nuclei Staining and Counting
Count each sample using a hemacytometer and microscope.
Note
A tutorial by Yevgeniy Grigoryev on using a hemacytometer and microscope for counting nuclei can be found at:
Add 10 µL sample and 10 µL AO/PI stain to a 0.5 mL tube. Pipet 10X to mix.
Load 10 µL of stained sample onto a hemacytometer and count nuclei for each sample. Record values, and assess clumping and integrity.
Following nuclei counting, proceed immediately to nuclei fixation using the Parse Evercode Nuclei Fixation Kit from Parse Biosciences.
Protocol references
Whytock, K. L., Divoux, A., Sun, Y., Hopf, M., Yeo, R. X., Pino, M. F., Yu, G., Smith, S. R., Walsh, M. J., & Sparks, L. M. (2023). Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling. STAR Protocols, 4(1), 102054. https://doi.org/10.1016/j.xpro.2023.102054