C. albicans haploinsufficiency profiling (HIP)

C. albicans haploinsufficiency (HIP) was performed as previously described.^11,21 Glycerol stock pools of heterozygous (HET) double-barcoded deletion mutants²⁰ were thawed, diluted to an OD₆₀₀ of 0.05 into a 60 mL YPD culture, and grown at 30°C under shaking conditions for 1.5 hours. Subsequently, 1 mL of the sub-cultured HET pool was aliquoted into triplicate culture tubes, each containing 1mL YPD medium with either 8 μg/mL (2X) NPD6433 or a DMSO solvent control. These cultures were grown at 30°C under shaking conditions for 18 hours. Cells were pelleted by centrifugation, the supernatant was removed, and cell pellets were stored at −80°C. Cell pellets were digested with Zymolyase in buffer (1 M sorbitol, 10 mM sodium EDTA, 14 mM β-mercaptoethanol, 15 units of Zymolyase enzyme) prior to genomic DNA extraction using the PureLink Genomic DNA Extraction kit, as per the manufacturer’s instructions (Invitrogen). Genomic DNA was recovered from columns provided by the kit using 10 mM Tris-HCl pH 8.0 and quantified using the PicoGreen DNA quantification kit (Invitrogen). Barcodes were PCR amplified with Takara Ex-Taq (Clonetech) using 150 ng of genomic DNA. UP-TAG primers (UP-TAG U and UP-TAG INX) and DOWN-TAG primers (DOWN-TAG U and DOWN-TAG INX) were used. The indexing primers contain a specific 6-base pair indexing sequence (See Table S3) to deconvolute treatment groups after sequencing. Equal quantities of UP-TAG and DOWN-TAG pools were combined to form a sequencing library, which was sequenced on an Illumina Next-Seq500 instrument (Mid-Output, V2 Chemistry) using specific primers to sequence and index the UP-(UP-TAG S and UP-TAG SINX) and DOWN-TAG (DOWN-TAG S and DOWN-TAG SINX) pools for each sample (see Table S3). Barcode-sequence reads were mapped to an artificial genome containing known UP-TAG and DOWN-TAG sequences of each strain and compiled for each indexed sample. If a specific UP-TAG or DOWN-TAG had more than one of its triplicate samples in the solvent control condition with read counts < 20% of the median read per million mapped, these reads were filtered and omitted from further analysis. Log₂ fold differences for each strain’s UP-TAG and DOWN-TAG were calculated. Significant outliers were identified as strains with a >25 median absolute deviations (MADs) in both the UP-TAG and DOWN-TAG, or if one of the UP-TAG and DOWN-TAG was >25 MADs and the opposing TAG was omitted due to low reads (UP-TAG 25X MAD = 1.9005, DOWN-TAG 25X MAD = 3.3125).