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.
Keywords: Next Generation Sequencing, pooled screens, RNA interference,
shRNA, validation.