Rapid creation and quantitative monitoring of high coverage shRNA libraries

Abstract

On-array synthesis of over 20,000 shRNAs at a coverage of ∼30 shRNAs per gene, followed by cloning into lentiviral shRNA libraries and deconvolution of the complex libraries by deep sequencing, ensures high confidence in the observed knockdown phenotypes with low false-negative rates and few off-target hits. Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (∼30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.