Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

Autor:	Göttert H; The Donnelly Centre, University of Toronto, 160 College St., Toronto, ON, M5S 3E1, Canada.; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, ON, M5S 3E1, Canada., Mattiazzi Usaj M; The Donnelly Centre, University of Toronto, 160 College St., Toronto, ON, M5S 3E1, Canada., Rosebrock AP; The Donnelly Centre, University of Toronto, 160 College St., Toronto, ON, M5S 3E1, Canada.; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, ON, M5S 3E1, Canada.; Department of Pathology and University Cancer Center, Stony Brook Medicine, Stony Brook, 11794, NY, USA., Andrews BJ; The Donnelly Centre, University of Toronto, 160 College St., Toronto, ON, M5S 3E1, Canada. brenda.andrews@utoronto.ca.; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, ON, M5S 3E1, Canada. brenda.andrews@utoronto.ca.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2018; Vol. 1672, pp. 613-629.
DOI:	10.1007/978-1-4939-7306-4_40
Abstrakt:	Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains. We also present an alternative pipeline for the analysis of R-SGA output strains using flow cytometry of cells in liquid culture. Data normalization for both pipelines is discussed.
Databáze:	MEDLINE
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