Generation of a pooled shRNA library for functional genomics screens.
| Autor: | Papadopoulos D; Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany., Ade CP; Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany., Eilers M; Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany. |
|---|---|
| Jazyk: | angličtina |
| Zdroj: | STAR protocols [STAR Protoc] 2022 Feb 15; Vol. 3 (1), pp. 101183. Date of Electronic Publication: 2022 Feb 15 (Print Publication: 2022). |
| DOI: | 10.1016/j.xpro.2022.101183 |
| Abstrakt: | Here, we detail a protocol for the generation of pooled short hairpin RNA (shRNA) libraries. We cover the design of optimized miR-E backbone shRNAs, cloning into a Tet-on vector system, and transformation of competent bacteria. We also describe library quality check by next-generation sequencing, and finally the production of lentiviruses. This protocol will generate high-quality inducible libraries suitable for both genome-wide and targeted functional genomics screens, allowing the high-throughput interrogation of protein depletion effects in the cell system of choice. For complete details on the use and execution of this protocol, please refer to Papadopoulos et al. (2022). Competing Interests: The authors declare no competing interests. (© 2022 The Author(s).) |
| Databáze: | MEDLINE |
| Externí odkaz: |
|