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

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

DOI

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.

OriginalsprogEngelsk
Artikelnummer2186686
TidsskriftNucleus
Vol/bind14
Nummer1
ISSN1949-1034
DOI
StatusUdgivet - dec. 2023

Bibliografisk note

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

Publisher Copyright:
© 2023 Aarhus Universitet. Published by Informa UK Limited, trading as Taylor & Francis Group.

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