title: Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens.
abstract: RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ∼10,000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.
authors.affiliation: Molecular Biology, Thermo Fisher Scientific, Lafayette, Colorado, United States of America.
authors.forename: Žaklina
authors.initials: Ž
authors.lastname: Strezoska
authors.forename: Abel
authors.initials: A
authors.lastname: Licon
authors.forename: Josh
authors.initials: J
authors.lastname: Haimes
authors.forename: Katie Jansen
authors.initials: KJ
authors.lastname: Spayd
authors.forename: Kruti M
authors.initials: KM
authors.lastname: Patel
authors.forename: Kevin
authors.initials: K
authors.lastname: Sullivan
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Jastrzebski
authors.forename: Kaylene J
authors.initials: KJ
authors.lastname: Simpson
authors.forename: Devin
authors.initials: D
authors.lastname: Leake
authors.forename: Anja
authors.initials: A
authors.lastname: van Brabant Smith
authors.forename: Annaleen
authors.initials: A
authors.lastname: Vermeulen
mesh_terms: D015723:Gene Library; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering