Pooled shRNA Screening
- Pp. 58-78 (21)Annaleen Vermeulen, Anja van Brabant Smith, Sarah B. Anderson, Roderick L. Beijersbergen and Kaylene J. Simpson
Short hairpin RNA interference (shRNA) screens have earned their place in the technical repertoire of high throughput screening approaches by virtue of their broad applicability to targeting regular and primary cell types and the capacity to perform both positive and negative selection screens both in vitro and in vivo. This chapter focuses primarily on pooled shRNA screens, outlining the breadth of resources available, important library features and methods to establish effective transduction. We discuss assay development and optimization, followed by strategies for hit identification, principally using Next Generation Sequencing (NGS) approaches. Validation of any screen is essential and our collective experience guides the reader to consider a range of approaches towards confirming targets identified in the screen subsequently recapitulate the biological premise of the screen. We conclude with a thought provoking discussion on the future of shRNA screens, the challenges and the scope we can look forward to.