Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies.
Nucleus
; 14(1): 2186686, 2023 12.
Article
in En
| MEDLINE
| ID: mdl-36878883
This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Urinary Bladder Neoplasms
Type of study:
Guideline
Limits:
Humans
Language:
En
Journal:
Nucleus
Year:
2023
Document type:
Article
Affiliation country:
Denmark
Country of publication:
United States