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Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies
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 an...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10012951/ https://www.ncbi.nlm.nih.gov/pubmed/36878883 http://dx.doi.org/10.1080/19491034.2023.2186686 |
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author | Schmøkel, Sofie S. Nordentoft, Iver Lindskrog, Sia V. Lamy, Philippe Knudsen, Michael Jensen, Jørgen Bjerggaard Dyrskjøt, Lars |
author_facet | Schmøkel, Sofie S. Nordentoft, Iver Lindskrog, Sia V. Lamy, Philippe Knudsen, Michael Jensen, Jørgen Bjerggaard Dyrskjøt, Lars |
author_sort | Schmøkel, Sofie S. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10012951 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-100129512023-03-15 Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies Schmøkel, Sofie S. Nordentoft, Iver Lindskrog, Sia V. Lamy, Philippe Knudsen, Michael Jensen, Jørgen Bjerggaard Dyrskjøt, Lars Nucleus Methods 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. Taylor & Francis 2023-03-06 /pmc/articles/PMC10012951/ /pubmed/36878883 http://dx.doi.org/10.1080/19491034.2023.2186686 Text en © 2023 Aarhus Universitet. Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Schmøkel, Sofie S. Nordentoft, Iver Lindskrog, Sia V. Lamy, Philippe Knudsen, Michael Jensen, Jørgen Bjerggaard Dyrskjøt, Lars Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
title | Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
title_full | Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
title_fullStr | Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
title_full_unstemmed | Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
title_short | Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
title_sort | improved protocol for single-nucleus rna-sequencing of frozen human bladder tumor biopsies |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10012951/ https://www.ncbi.nlm.nih.gov/pubmed/36878883 http://dx.doi.org/10.1080/19491034.2023.2186686 |
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