Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
STAR Protoc. 2022 Feb 15;3(1):101183. doi: 10.1016/j.xpro.2022.101183. eCollection 2022 Mar 18.
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).
在这里，我们详细介绍了一种用于生成 pooled short hairpin RNA (shRNA) 文库的方案。我们涵盖了优化的 miR-E 骨干 shRNA 的设计、克隆到 Tet-on 载体系统，以及感受态细菌的转化。我们还描述了通过下一代测序进行文库质量检查，以及最后制备慢病毒。该方案将生成高质量的诱导文库，适用于全基因组和靶向功能基因组筛选，允许在所选细胞系统中高通量检测蛋白质缺失效应。有关该方案的使用和执行的完整详细信息，请参阅 Papadopoulos 等人 (2022)。
