Fig. 2

a Deletion strain strategy. The KanMX gene expressed from the TEF1 promoter confers dominant selection of geneticin (G418) to yeast and was amplified with primers adding common primers (orange), molecular barcodes or tags (violet), and homologies with the chromosome (maroon). When transformed with the linear PCR product, cells replace the targeted ORF with the KanMX cassette flanked with UPtag and DOWNtag using homologous recombination. b Description of the competitive growth assay. Fitness profiling of pooled deletion strains involves six main steps. (1) Strains are first pooled at approximately equal abundance. (2) The pool is grown competitively in the condition of choice. If a gene is required for growth under this condition, the strain carrying this deletion will grow more slowly and become underrepresented in the culture (orange strain) over time. Similarly, resistant strains will grow faster and become overrepresented (maroon strain). (3) Genomic DNA is isolated from cells is harvested at the end of pooled growth. (4) Barcodes are amplified from the genomic DNA with universal primers in two PCR reactions, one for the uptags and one for the downtags. (5) Resulting PCR products are sequenced by NGS to quantify the tag sequences and, therefore, the relative abundance of each strain. (6) Tag intensities for the treatment sample are compared to tag intensities for a control sample to determine the relative fitness of each strain