title: A comprehensive platform for highly multiplexed mammalian functional genetic screens.
abstract: BACKGROUND
  Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.
  
  
  RESULTS
  Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.
  
  
  CONCLUSIONS
  Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.
authors.affiliation: Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada.
authors.forename: Troy
authors.initials: T
authors.lastname: Ketela
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Heisler
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Brown
authors.forename: Ron
authors.initials: R
authors.lastname: Ammar
authors.forename: Dahlia
authors.initials: D
authors.lastname: Kasimer
authors.forename: Anuradha
authors.initials: A
authors.lastname: Surendra
authors.forename: Elke
authors.initials: E
authors.lastname: Ericson
authors.forename: Kim
authors.initials: K
authors.lastname: Blakely
authors.forename: Dina
authors.initials: D
authors.lastname: Karamboulas
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Smith
authors.forename: Tanja
authors.initials: T
authors.lastname: Durbic
authors.forename: Anthony
authors.initials: A
authors.lastname: Arnoldo
authors.forename: Kahlin
authors.initials: K
authors.lastname: Cheung-Ong
authors.forename: Judice L Y
authors.initials: JL
authors.lastname: Koh
authors.forename: Shuba
authors.initials: S
authors.lastname: Gopal
authors.forename: Glenn S
authors.initials: GS
authors.lastname: Cowley
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Yang
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Grenier
authors.forename: Guri
authors.initials: G
authors.lastname: Giaever
authors.forename: David E
authors.initials: DE
authors.lastname: Root
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
mesh_terms: D000818:Animals; D005820:Genetic Testing / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames / Q000235:genetics; D011786:Quality Control; D034622:RNA Interference; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012984:Software