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).