Figure S2.
Construction and characterization of genome-wide AID libraries. (a) Fitness impact of OsTIR1 expression from different promoters. 10-fold serial dilutions of yeast strains on the indicated media without 5-Ph-IAA. (b) Evaluation of the SWAT tagging efficiency during construction of the AID libraries. For the AID-v2 library, control tagging was performed with 13 C-SWAT strains for the indicated ORFs and a donor with the mCherry-sfGFP timer (tFT) as a tag. Distributions of single-cell mNG fluorescence intensities measured with flow cytometry (10,000 cells per strain). The percentage of cells with fluorescence above background (fluorescence of a wild type strain, dashed line) is indicated. (c) Correlation between absolute protein abundance (Lawless et al., 2016) and relative protein expression levels measured with the AID-v1 library lacking OsTIR1. Fluorescence intensities of colonies were corrected for background fluorescence. Linear fit (dashed line) and estimate of the detection limit of the colony assay (200 molecules/cell) with the 95% confidence interval (18 to 2,187 molecules/cell). (d) Comparison of relative protein expression levels measured with the AID-v1 library lacking OsTIR1 and with the mNG-II library (ORF-mNG strains) (Meurer et al., 2018). Fluorescence intensities of colonies were corrected for background fluorescence. r, Pearson correlation coefficient. (e) Degradation of AID-tagged proteins after addition of 5-Ph-IAA. Whole-cell extracts of OsTIR1+ strains for the indicated proteins (AID-v1 library, left) were separated by SDS-PAGE, followed by immunoblotting with antibodies against the myc tag and Pgk1 as the loading control (right). (f) Distribution of background-corrected mNG fluorescence intensities in the AID-v1 library, OsTIR1− strains, determined according to Fig. 2 c. Number of proteins in the not detected group (mNG/bkg(OsTIR1−) ≤ 1.2) and in five abundance bins used in Fig. 2 e; Fig. 3 d; Fig. S3 b; and Fig. S4 e is indicated. Source data are available for this figure: SourceData FS2.

Construction and characterization of genome-wide AID libraries. (a) Fitness impact of OsTIR1 expression from different promoters. 10-fold serial dilutions of yeast strains on the indicated media without 5-Ph-IAA. (b) Evaluation of the SWAT tagging efficiency during construction of the AID libraries. For the AID-v2 library, control tagging was performed with 13 C-SWAT strains for the indicated ORFs and a donor with the mCherry-sfGFP timer (tFT) as a tag. Distributions of single-cell mNG fluorescence intensities measured with flow cytometry (10,000 cells per strain). The percentage of cells with fluorescence above background (fluorescence of a wild type strain, dashed line) is indicated. (c) Correlation between absolute protein abundance (Lawless et al., 2016) and relative protein expression levels measured with the AID-v1 library lacking OsTIR1. Fluorescence intensities of colonies were corrected for background fluorescence. Linear fit (dashed line) and estimate of the detection limit of the colony assay (200 molecules/cell) with the 95% confidence interval (18 to 2,187 molecules/cell). (d) Comparison of relative protein expression levels measured with the AID-v1 library lacking OsTIR1 and with the mNG-II library (ORF-mNG strains) (Meurer et al., 2018). Fluorescence intensities of colonies were corrected for background fluorescence. r, Pearson correlation coefficient. (e) Degradation of AID-tagged proteins after addition of 5-Ph-IAA. Whole-cell extracts of OsTIR1+ strains for the indicated proteins (AID-v1 library, left) were separated by SDS-PAGE, followed by immunoblotting with antibodies against the myc tag and Pgk1 as the loading control (right). (f) Distribution of background-corrected mNG fluorescence intensities in the AID-v1 library, OsTIR1− strains, determined according to Fig. 2 c. Number of proteins in the not detected group (mNG/bkg(OsTIR1−) ≤ 1.2) and in five abundance bins used in Fig. 2 e; Fig. 3 d; Fig. S3 b; and Fig. S4 e is indicated. Source data are available for this figure: SourceData FS2.

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