Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies

ABSTRACT

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.

KEYWORDS:

• 10× chromium
• bladder cancer
• Drop-seq
• DroNc-seq
• nuclei isolation
• single-cell analysis
• single-cell RNA-sequencing
• single-nuclei RNA-sequencing
• single-nucleus RNA-sequencing

Acknowledgments

We would like to thank all technical personnel at the Department of Molecular Medicine and Department of Urology and Oncology, Aarhus University Hospital, for sample handling and processing. We would like to thank GenomeDK and Aarhus University for providing computational resources that contributed to these research results.

Disclosure statement

Lars Dyrskjøt has sponsored research agreements with C2i, AstraZeneca, Natera, Photocure, and Ferring; has an advisory/consulting role at Ferring and UroGen; and is Chairman of the Board in BioXpedia A/S. Jørgen Bjerggaard Jensen is proctor for Intuitive Surgery; is a member of advisory board for Olympus Europe, Ambu, Cepheid, and Ferring; and has sponsored research agreements with Medac, Photocure ASA, Cepheid, and Ferring.

Author contributions

Conceptualization: S.S.S., I.N., L.D.; data curation: S.S.S., S.V.L., P.L., M.K.; formal analysis: S.S.S., S.V.L., P.L.; funding acquisition: L.D.; investigation: S.S.S., I.N.; methodology: S.S.S., I.N., L.D.; project administration: L.D.; resources: J.B.J., L.D.; supervision: L.D.; visualization: S.S.S., M.K.; writing – original draft: S.S.S., I.N., S.V.L., L.D.; writing – review and editing: all authors.

Data availability statement

The raw sequencing data generated in this study are not publicly available as these compromise patient consent and ethics regulations in Denmark. Processed nonsensitive data are available upon reasonable request from the corresponding author.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19491034.2023.2186686

Additional information

Funding

Independent Research Fund Denmark, The Novo Nordisk Foundation, Aarhus University (AUFF NOVA), The Leo & Anne Albert Institute for Bladder Cancer Care and Research.