Ready for your all-in-one single cell sequencing solution?
Contact us

How to Isolate Nuclei for Single Cell Methods 

5’
Protocol and guide

Single nucleus analysis is essential for single cell studies of hard-to-dissociate or frozen tissues, and enables multi-omics assays such as scATAC-seq. However, nuclei isolation is technically demanding and requires careful optimisation.  

This guide consolidates current best practices, peer-reviewed evidence, and hands-on tips to help you extract intact, transcriptionally faithful nuclei—no matter the tissue type. 

What is nuclei isolation? 

Nuclei house genomic DNA and a sizeable fraction of nascent, unspliced pre-mRNA, making them rich reservoirs of transcriptomic and regulatory information (Bakken et al., 2018). 
Nuclei isolation is the controlled disruption of the plasma membrane while preserving the nuclear envelope, yielding a suspension of intact nuclei for downstream assays such as: 

  • snRNA-seq (single nucleus RNA sequencing) 
  • scATAC-seq (single cell ATAC sequencing) 
  • Multi-omics (e.g., RNA + Chromatin accessibility from the same nucleus) 

Dive deeper: A Guide to scATAC-seq

Read more

When do you need nuclei isolation? 

  1. Difficult-to-dissociate tissues: adipose, muscle, heart, brain, and fibrotic tissues often challenging to dissociate into single cells. 
  1. Frozen tissues: flash-frozen specimens cannot be dissociated into viable single cells, but nuclei remain accessible. 
  1. scATAC-seq interrogates chromatin accessibility and requires single nuclei as the starting material. 

Unsure whether to run single-cell or single-nucleus?

Download our decision white paper

Preparation for nuclei isolation

Nuclei are sensitive to RNAse activity. Before tissue processing, treat the bench and tools with RNaseZap or similar solutions, then wipe them down with nuclease-free water. This minimises RNase contamination, preserving transcript integrity. 

For organoids or 3-D cultures, remove extracellular matrix to avoid it interfering with nuclei isolation. 

Key methods of cell lysis and nuclei isolation 

Cell lysis is the most variable and crucial step in nuclei isolation. The goal is to permeabilise the plasma membrane while leaving the nuclear envelope intact. 

Optimisation is important as each sample type can behave differently. Key parameters include: 

  • Lysis buffer: usually a mix of detergents and enzymes. Detergents such as Triton X-100 are commonly used (Nadelmann et al., 2022; Sikorskaite et al., 2013; Wiegleb et al., 2022). Both home-brew reagents and commercial kits are available. Different sample types may require different buffers. 
  • Mechanical agitation: Mechanical cutting, shearing, and homogenisation complement enzymatic treatment, helping to break up large tissue pieces and reduce reaction time. This ranges from gentle inversion and pipetting to Dounce homogenisation 
  • Time: Typically, the lysis time can be between 1 to 10 minutes.  

Add RNase inhibitor to protect RNA during processing. 

It is recommended to regularly check the status of cell lysis. Here are some examples of good quality nuclei versus overly lysed samples:

Single, round nuclei with sharp borders
Blebbing. Ruptured nucleus membrane

Mix 10 µL nuclei suspension 1:1 with Trypan Blue or Propidium Iodide (PI).
Good: ≥90 % single, round nuclei with sharp borders.
Over-lysed: Blebs, DNA halos, or ruptured envelopes.
Under-lysed: Most cells still remain intact.

Singleron’s PythoN I and PythoN Junior automate the nuclei isolation procedure, ensuring consistent output for every run. With a fully adjustable programme, they combine the advantages of full automation and the flexibility to fine-tune parameters for your specific samples.

Should I use a nuclei isolation kit? 

  • Many labs have their established nuclei isolation protocols tailored to their tissue of interest. 
  • For labs that are establishing protocols for the first time, pre-validated commercial kits can reduce optimisation time and reduce batch-to-batch variations. 
  • Fatty samples (e.g., adipose tissue) often require specific kits for high lipid content.

Singleron nuclei isolation kits have been validated across a wide range of tissue types. 

Contact us to discuss your specific species or tissue

Nuclei purification and quality control

  • After nuclei isolation, the resulting nuclei suspension is washed multiple times by centrifugation. 
  • Nuclei can be stained using PI, Trypan blue, or Acridine Orange/PI (AOPI) for integrity check. 
  • Accurate counting is important for downstream processing and single cell experiment. Manual counting is recommended. 

Additional tips

Pilot first

Even identical tissue types behave differently across donors or post-mortem intervals.

Flash-freezing best practice

Place intact pieces of tissue in a microfuge or cryo tube. Immerse the tube in liquid N₂ immediately; avoid pre-chopping to reduce surface ice crystal damage. .

Sample size

5–30 mg optimises lysis kinetics—too little yields scant nuclei, too much risks incomplete permeabilisation.

Anti-clumping

0.5–1 % BSA in all wash buffers and nuclei resuspension buffers.

Live QC loop

Inspect a 5 µL aliquot every 1-2 min during lysis when optimising a new protocol.

Conclusion

High-quality nuclei isolation demands a delicate balance between efficient cell membrane disruption and preservation of nuclear integrity. By systematically tuning detergent strength, lysis timing, and mechanical force—backed by rigorous QC—you will maximise usable nuclei and data quality in snRNA-seq, scATAC-seq, and multi-omics workflows.

Need hands-on support? Singleron’s applications team offers sample-specific protocol optimization and full-service nuclei isolations.

Contact a Singleron scientist

Reference: 

Bakken, T. E., Hodge, R. D., Miller, J. A., et al. (2018). Single-nucleus and single-cell transcriptomes compared in matched cortical cell types. PLOS ONE 13(12): e0209648. https://doi.org/10.1371/journal.pone.0209648 

Wiegleb, G., Reinhardt, S., Dahl, A. et al. (2022). Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material. Frontiers in Zoology, 19(1), 27. https://doi.org/10.1186/s12983-022-00472-x

Nadelmann, E. R., Gorham, J. M., Reichart, D. et al. (2021). Isolation of nuclei from mammalian cells and tissues for single-nucleus molecular profiling. Current Protocols, 1(5), e132. https://doi.org/10.1002/cpz1.132

Sikorskaite, S., Rajamäki, M. L., Baniulis, D. et al. (2013). Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families. Plant Methods, 9, 31. https://doi.org/10.1186/1746-4811-9-31

A post by Ray Wang

Ray is a senior applications specialist at Singleron Biotechnologies. Ray has 7 years of experience in single cell multi-omics, and is highly experienced in tissue processing and sample preparation. You can say Ray is our single cell knowledge base and our go-to-person for all practical single cell tips. Besides his wet lab expertise, Ray has also built multiple apps to assist with single cell wet lab workflow. In his free time, he has been a certified kayaking coach for the past 13 years.

Check out our latest blog posts

Learn more

What is Bulk RNA Sequencing?

The basics of bulk RNA sequencing Bulk RNA sequencing (bulk RNA-seq) is a powerful transcriptomic tool that measures gene expression across a pooled population of…

Read more