Should I use whole cells or nuclei? When is nuclei isolation required?

Answer:
Whole cells are generally recommended, as they retain both nuclear and cytoplasmic transcripts.

However, nuclei isolation is recommended under the following conditions:

• Cells are large, fragile, or difficult to dissociate 
• Samples have been frozen 
• Tissue structure limits effective cell recovery 

It should be noted that nuclei-based workflows capture primarily nuclear RNA and therefore lose cytoplasmic transcripts. In addition, nuclei are not suitable for V(D)J analysis, due to the absence of cytoplasmic mRNA.

Recommended sample types for nuclei isolation:

Large or fragile cell types:

• Liver (hepatocytes, 20–40 μm) 
• Pancreas (acinar cells) 
• Brain (neurons) 
• Adipose tissue (mature adipocytes) 
• Heart (cardiomyocytes) 
• Muscle (skeletal muscle cells) 
• Large protoplasts (>35 μm) 

These cells are prone to rupture during dissociation and may lead to increased background and empty droplets.

Frozen tissues:
Only nuclei can be used due to loss of cell viability. 

Delayed experimental confirmation scenarios:
When immediate processing is not possible (e.g., pending pathology results), tissues may be frozen and processed later via nuclei isolation. 

Nuclei suspension requirements:

• Buffer: 1× PBS (Ca²⁺/Mg²⁺-free) 

Additives: RNase inhibitor (e.g., Sigma-Aldrich #3335402001; final concentration 1 U/μL) + 0.1% BSA