High-Throughput Protein Localization Screening

Four clones at least were picked from each transformation. Validation PCR was performed using a common forward primer from the 3′ end of the SWAT modules (S4 reverse complement), and a gene specific reverse primer from the gene coding sequence. All four clones were imaged via a high-content screening platform in bright-field and GFP channels (see below). Images of all clones were reviewed manually, and up to three localizations were assigned to each clone. Assignments were: Ambiguous; Below threshold; Bud; Bud neck; Cell periphery; Cytosol; ER; Mitochondria; Nuclear periphery; Nucleolus; Nucleus; Punctate; Vacuole; Vacuole membrane. Only strains with a duplicate repeating localization assignment and validated by PCR were chosen to compose the SWAT-GFP and SWAP-SP-GFP collections.

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Yofe I., Weill U., Meurer M., Chuartzman S., Zalckvar E., Goldman O., Ben-Dor S., Schütze C., Wiedemann N., Knop M., Khmelinskii A, & Schuldiner M. (2016). One library to make them all: Streamlining yeast library creation by a SWAp-Tag (SWAT) strategy. Nature methods, 13(4), 371-378.

Publication 2016

A gene Cell Clone Coding sequence Cytosol Membrane Mitochondria Neck Nucleolus Nucleus Primer Strains Vacuole

Corresponding Organization : Weizmann Institute of Science

Other organizations : DKFZ-ZMBH Alliance, Heidelberg University

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Protocol cited in 13 other protocols

Variable analysis

independent variables

Number of clones picked from each transformation

dependent variables

Localization of clones (Ambiguous, Below threshold, Bud, Bud neck, Cell periphery, Cytosol, ER, Mitochondria, Nuclear periphery, Nucleolus, Nucleus, Punctate, Vacuole, Vacuole membrane)

control variables

Common forward primer from the 3' end of the SWAT modules (S4 reverse complement)
Gene specific reverse primer from the gene coding sequence
High-content screening platform in bright-field and GFP channels
Manual review of clone images
Requirement of duplicate repeating localization assignment and PCR validation for inclusion in SWAT-GFP and SWAP-SP-GFP collections

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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