Generation of a pooled shRNA library for functional genomics screens

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). Keywords: Bioinformatics; Genomics; High Throughput Screening; Molecular Biology; Sequence analysis; Sequencing.