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An analysis and validation pipeline for large-scale RNAi-based screens

Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress r...

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Autores principales: Plank, Michael, Hu, Guang, Silva, A. Sofia, Wood, Shona H., Hesketh, Emily E., Janssens, Georges, Macedo, André, de Magalhães, João Pedro, Church, George M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546318/
https://www.ncbi.nlm.nih.gov/pubmed/23326633
http://dx.doi.org/10.1038/srep01076
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author Plank, Michael
Hu, Guang
Silva, A. Sofia
Wood, Shona H.
Hesketh, Emily E.
Janssens, Georges
Macedo, André
de Magalhães, João Pedro
Church, George M.
author_facet Plank, Michael
Hu, Guang
Silva, A. Sofia
Wood, Shona H.
Hesketh, Emily E.
Janssens, Georges
Macedo, André
de Magalhães, João Pedro
Church, George M.
author_sort Plank, Michael
collection PubMed
description Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress resistance and found several candidates. We then developed a bioinformatics pipeline to prioritize these candidates incorporating effect sizes, functional enrichment analysis, interaction networks and gene expression information. To validate candidates, we mixed normal cells with cells expressing the shRNA coupled to a fluorescent protein, which allows control cells to be used as an internal standard, and thus we could detect shRNAs with subtle effects. Although we did not identify genes associated with oxidative stress resistance, as a proof-of-concept of our pipeline we demonstrate a detrimental role of Edd1 silencing in ESC growth. Our methods may be useful for candidate gene prioritization of large-scale RNAi-based screens.
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spelling pubmed-35463182013-01-16 An analysis and validation pipeline for large-scale RNAi-based screens Plank, Michael Hu, Guang Silva, A. Sofia Wood, Shona H. Hesketh, Emily E. Janssens, Georges Macedo, André de Magalhães, João Pedro Church, George M. Sci Rep Article Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress resistance and found several candidates. We then developed a bioinformatics pipeline to prioritize these candidates incorporating effect sizes, functional enrichment analysis, interaction networks and gene expression information. To validate candidates, we mixed normal cells with cells expressing the shRNA coupled to a fluorescent protein, which allows control cells to be used as an internal standard, and thus we could detect shRNAs with subtle effects. Although we did not identify genes associated with oxidative stress resistance, as a proof-of-concept of our pipeline we demonstrate a detrimental role of Edd1 silencing in ESC growth. Our methods may be useful for candidate gene prioritization of large-scale RNAi-based screens. Nature Publishing Group 2013-01-16 /pmc/articles/PMC3546318/ /pubmed/23326633 http://dx.doi.org/10.1038/srep01076 Text en Copyright © 2013, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by/3.0/ This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/
spellingShingle Article
Plank, Michael
Hu, Guang
Silva, A. Sofia
Wood, Shona H.
Hesketh, Emily E.
Janssens, Georges
Macedo, André
de Magalhães, João Pedro
Church, George M.
An analysis and validation pipeline for large-scale RNAi-based screens
title An analysis and validation pipeline for large-scale RNAi-based screens
title_full An analysis and validation pipeline for large-scale RNAi-based screens
title_fullStr An analysis and validation pipeline for large-scale RNAi-based screens
title_full_unstemmed An analysis and validation pipeline for large-scale RNAi-based screens
title_short An analysis and validation pipeline for large-scale RNAi-based screens
title_sort analysis and validation pipeline for large-scale rnai-based screens
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546318/
https://www.ncbi.nlm.nih.gov/pubmed/23326633
http://dx.doi.org/10.1038/srep01076
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