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RNAi Screening with Pooled Lentiviral shRNA Libraries

Cellecta Strategy. Focused and genome-wide lentiviral pooled bar-coded shRNA libraries (pre-designed and customized)HT RNAi Genetic Screens w/ pooled bar-coded lentiviral shRNA librariesReporter cell line development for genetic screensBioinformatic data analysis for RNAi Genetic ScreensCell-based HTS for bioactive peptides w/ pooled peptide libraries Rational Directed Evolution for peptide lead optimization.

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RNAi Screening with Pooled Lentiviral shRNA Libraries

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    1. RNAi Screening with Pooled Lentiviral shRNA Libraries

    2. Cellecta Strategy Focused and genome-wide lentiviral pooled bar-coded shRNA libraries (pre-designed and customized) HT RNAi Genetic Screens w/ pooled bar-coded lentiviral shRNA libraries Reporter cell line development for genetic screens Bioinformatic data analysis for RNAi Genetic Screens Cell-based HTS for bioactive peptides w/ pooled peptide libraries Rational Directed Evolution for peptide lead optimization

    3. Tools for RNAi Screens

    4. Genetic Screen Formats

    5. Arrayed vs Pooled siRNA/shRNA Libraries

    6. Pooled shRNA Library Screening

    9. Lessons from RNAi Screens Design of biological assay – at least 50 to 100-fold enrichment of cells with a specific phenotype in one round of selection Reduction of false negative rate – use second-generation validated libraries with redundant sets of shRNAs per gene combined with HT sequencing - significant improvement in quality of data over microarray hybridization Reduction of false positive rate – use reduced-complexity focused shRNA libraries with 100-300-fold excess of cells in the screen above the complexity of the library Confirmation of hits – second round of screening with enriched sublibrary, biological triplicates, knockdown with specific shRNA constructs, correlation with pathway analysis programs, and expression profiling data

    10. Fluorescence Reporter Assay for shRNA Validation

    11. HT Functional shRNA Screening Strategy

    12. Percentage of Functional shRNAs Percentage of shRNA that knock down target mRNA >70%, based on knockdown activity of 120 shRNA constructs targeting 8 genes:

    13. Positive and Negative Selection Screens with Second Generation Libraries PUMA-induced apoptosis in HeLa cells TGF-? induced apoptosis in Hep3B cells Viability of K-562 cells

    15. Comparison of PUMA-induced apoptosis screens using shRNA libraries with HT sequencing or Microarray hybridization

    17. Signaling Pathways Identified by TGF-? Screening Red ? : by Cellecta’s 25K HT Seq shRNA library Green ? : by Cellecta’s 1st Generation library Blue ? : other commercial library Data analysis using Ingenuity Pathway Analysis Software.

    20. Discover Cancer Drug Targets

    21. Design of shRNA Libraries

    22. Andrei Gudkov – Roswell Park Cancer Institute Costas G. Frangou – FHCRC Peiqing Sun – The Scripps Research Institute Lester Kobzik – Harvard School of Public Health Gary Sahagian – Tufts University Luda Diatchenko – University of North Carolina at Chapel Hill Yutaka Eguchi – Osaka University Acknowledgments

    23. Cellecta Contact Information

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