Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies
ABSTRACTThis 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 t...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2023-12-01
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| Series: | Nucleus |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/19491034.2023.2186686 |
| _version_ | 1797387378616172544 |
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| author | Sofie S. Schmøkel Iver Nordentoft Sia V. Lindskrog Philippe Lamy Michael Knudsen Jørgen Bjerggaard Jensen Lars Dyrskjøt |
| author_facet | Sofie S. Schmøkel Iver Nordentoft Sia V. Lindskrog Philippe Lamy Michael Knudsen Jørgen Bjerggaard Jensen Lars Dyrskjøt |
| author_sort | Sofie S. Schmøkel |
| collection | DOAJ |
| description | ABSTRACTThis 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. |
| first_indexed | 2024-03-08T22:23:16Z |
| format | Article |
| id | doaj.art-2e7747d78fe2400793b4ae335a10b1b4 |
| institution | Directory Open Access Journal |
| issn | 1949-1034 1949-1042 |
| language | English |
| last_indexed | 2024-03-08T22:23:16Z |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Nucleus |
| spelling | doaj.art-2e7747d78fe2400793b4ae335a10b1b42023-12-18T11:21:33ZengTaylor & Francis GroupNucleus1949-10341949-10422023-12-0114110.1080/19491034.2023.2186686Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsiesSofie S. Schmøkel0Iver Nordentoft1Sia V. Lindskrog2Philippe Lamy3Michael Knudsen4Jørgen Bjerggaard Jensen5Lars Dyrskjøt6Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkDepartment of Molecular Medicine, Aarhus University Hospital, Aarhus, DenmarkDepartment of Clinical Medicine, Aarhus University, Aarhus, DenmarkDepartment of Molecular Medicine, Aarhus University Hospital, Aarhus, DenmarkDepartment of Molecular Medicine, Aarhus University Hospital, Aarhus, DenmarkDepartment of Clinical Medicine, Aarhus University, Aarhus, DenmarkDepartment of Clinical Medicine, Aarhus University, Aarhus, DenmarkABSTRACTThis 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.https://www.tandfonline.com/doi/10.1080/19491034.2023.218668610× chromiumbladder cancerDrop-seqDroNc-seqnuclei isolationsingle-cell analysis |
| spellingShingle | Sofie S. Schmøkel Iver Nordentoft Sia V. Lindskrog Philippe Lamy Michael Knudsen Jørgen Bjerggaard Jensen Lars Dyrskjøt Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies Nucleus 10× chromium bladder cancer Drop-seq DroNc-seq nuclei isolation single-cell analysis |
| 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 | 10× chromium bladder cancer Drop-seq DroNc-seq nuclei isolation single-cell analysis |
| url | https://www.tandfonline.com/doi/10.1080/19491034.2023.2186686 |
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