Protocol for Comprehensive Synthetic Lethality Screens

• Published in: STAR protocols Vol. 1; no. 1; p. 100016
• Main Authors: Romero-Moya, Damia, Roose, Jeroen P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.06.2020; Elsevier
• Subjects: Gene Library; Humans; Indazoles; Jurkat Cells; Phosphoinositide-3 Kinase Inhibitors; Protocol; RNA, Small Interfering; Sulfonamides; Toxicity Tests - methods
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100016

Here, we provide a detailed protocol for synthetic lethality screens in a Jurkat T cell leukemia line using cell death as the readout measuring the combinatorial effect of a pan-PI3K inhibitor (GDC0941) with specific gene depletion by shRNA. We describe the use of an ultra-complex shRNA library, coverage considerations, time frames, protocol details, and bottlenecks with images to facilitate similar approaches. We discuss how this protocol resource can be readily adapted by investigators. For complete details on the use and execution of this protocol, please refer to (Mues et al., 2019). [Display omitted] •A protocol for synthetic lethality screens in non-adherent cells•Considerations and background information to consider before starting•Step-by-step protocol details with example images for easy application Here, we provide a detailed protocol for synthetic lethality screens in a Jurkat T cell leukemia line using cell death as the readout measuring the combinatorial effect of a pan-PI3K inhibitor (GDC0941) with specific gene depletion by shRNA. We describe the use of an ultra-complex shRNA library, coverage considerations, time frames, protocol details, and bottlenecks with images to facilitate similar approaches. We discuss how this protocol resource can be readily adapted by investigators.