title: Development of methods for quantitative comparison of pooled shRNAs by mass sequencing.
abstract: Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments.
authors.affiliation: Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390-9039, USA.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Hoshiyama
authors.forename: Jun
authors.initials: J
authors.lastname: Tang
authors.forename: Kimberly
authors.initials: K
authors.lastname: Batten
authors.forename: Guanghua
authors.initials: G
authors.lastname: Xiao
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Rouillard
authors.forename: Jerry W
authors.initials: JW
authors.lastname: Shay
authors.forename: Yang
authors.initials: Y
authors.lastname: Xie
authors.forename: Woodring E
authors.initials: WE
authors.lastname: Wright
mesh_terms: D002460:Cell Line; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D012313:RNA; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000032:analysis* / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: C034477:RNA primers; D034741:RNA, Small Interfering; D012313:RNA