دورية أكاديمية
Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies
| العنوان: | Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies |
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| المؤلفون: | Sofie S. Schmøkel, Iver Nordentoft, Sia V. Lindskrog, Philippe Lamy, Michael Knudsen, Jørgen Bjerggaard Jensen, Lars Dyrskjøt |
| المصدر: | Nucleus, Vol 14, Iss 1 (2023) |
| بيانات النشر: | Taylor & Francis Group, 2023. |
| سنة النشر: | 2023 |
| المجموعة: | LCC:Genetics LCC:Cytology |
| مصطلحات موضوعية: | 10× chromium, bladder cancer, Drop-seq, DroNc-seq, nuclei isolation, single-cell analysis, Genetics, QH426-470, Cytology, QH573-671 |
| الوصف: | 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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 19491034 1949-1042 1949-1034 |
| Relation: | https://doaj.org/toc/1949-1034; https://doaj.org/toc/1949-1042 |
| DOI: | 10.1080/19491034.2023.2186686 |
| URL الوصول: | https://doaj.org/article/2e7747d78fe2400793b4ae335a10b1b4 |
| رقم الأكسشن: | edsdoj.2e7747d78fe2400793b4ae335a10b1b4 |
| قاعدة البيانات: | Directory of Open Access Journals |
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Full Text Finder | تدمد: | 19491034 19491042 |
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| DOI: | 10.1080/19491034.2023.2186686 |