2017
DOI: 10.1101/226811
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A genome-wide resource for high-throughput genomic tagging of yeast ORFs

Abstract: Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae ORFs. It consists of 5661 strains with an acceptor module inserted after each ORF, which can be efficiently replaced with tags or regulatory elements. We validate the library with targeted sequencing and demonstrate its use by tagging the yeast proteome with bright fluorescent proteins, determining how sequences downstream of ORFs influence protein expression and localizing previously undetected proteins.

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Cited by 4 publications
(8 citation statements)
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“…Assignment categories were: Bud, Bud Neck, Cell Periphery, Cytosol, ER, Mitochondria, Nuclear Periphery, Nucleolus, Nucleus, Punctate, Vacuole and Vacuole Membrane. (iii) Determination of the SWAT swapping capability (See “Analysis of swapping-procedure efficiency” section); and finally (iv) Sequencing (Anchor-seq) to ensure correct reading frame: We employed a targeted-sequencing strategy detailed in 7 to verify the junction encompassing the 3’ end of the cassette and the 5’ end of each gene. Briefly, we pooled all strains from the SWAT library together, extracted their genomic DNA, sheared it into fragments of 300-800bp that were gel-purified, ligated to Anchor-seq adaptors and submitted to two rounds of PCR.…”
Section: Methodsmentioning
confidence: 99%
See 2 more Smart Citations
“…Assignment categories were: Bud, Bud Neck, Cell Periphery, Cytosol, ER, Mitochondria, Nuclear Periphery, Nucleolus, Nucleus, Punctate, Vacuole and Vacuole Membrane. (iii) Determination of the SWAT swapping capability (See “Analysis of swapping-procedure efficiency” section); and finally (iv) Sequencing (Anchor-seq) to ensure correct reading frame: We employed a targeted-sequencing strategy detailed in 7 to verify the junction encompassing the 3’ end of the cassette and the 5’ end of each gene. Briefly, we pooled all strains from the SWAT library together, extracted their genomic DNA, sheared it into fragments of 300-800bp that were gel-purified, ligated to Anchor-seq adaptors and submitted to two rounds of PCR.…”
Section: Methodsmentioning
confidence: 99%
“…In a short time period and at a fraction of the cost incurred to date using other approaches, any yeast laboratory can make its very own library harboring a diversity of selection markers, promoters, untranslated regions, targeting signals, fluorophores, affinity tags or any other genetic element of choice. Using this platform, the systematic exploration of any protein is no longer restricted and can be done either with N’ or C’ tagging 7. Together, these approaches should contribute greatly to our knowledge on how the living cell works.…”
Section: Perspectivesmentioning
confidence: 99%
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“…The mitochondrial-localized proteins of the budding yeast Saccharomyces cerevisiae, a model of eukaryotic cells, are examples of proteins that have been extensively investigated by such approaches. Proteins in fractionated mitochondria have been systematically identified by mass spectrometry (Morgenstern et al, 2017;Reinders et al, 2006;Sickmann et al, 2003;Vögtle et al, 2017) and the subcellular localization of fluorescent protein fusion proteins has been analyzed by fluorescence microscopy for nearly all proteins (Breker et al, 2013;Chong et al, 2015;Huh et al, 2003;Koh et al, 2015;Meurer et al, 2018;Weill et al, 2018). A complementary approach to the experimental approach is the prediction of localization by bioinformatics.…”
Section: Introductionmentioning
confidence: 99%
“…It is possible that there are further affected proteins which would only be revealed if a stronger overexpression was employed. Furthermore, since many proteins only tolerate either an N-or a C-terminal fusion to a tag without being functionally perturbed, a similar screen with a C-terminally GFP-tagged library may reveal even further hits (Meurer et al, 2018). Nevertheless, for TA proteins, the N-terminal fusion library that I screened is ideally suited since C-terminal GFP fusion abolishes the TA topology.…”
Section: Direct and Indirect Effects Of Loss Of Get3mentioning
confidence: 99%