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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2013
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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. |
format | Online Article Text |
id | pubmed-3546318 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
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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