96 well replicator

To assay the growth kinetics upon drug treatment, the wild-type strain was transferred via 96-well replicator (Singer Instruments) from an overnight culture into 100 μL of YEPD with the indicated concentration of fluconazole (Sequoia Research Products Ltd SRP01025f), terbinafine (Sigma T8826), amphotericin B (Sigma A4888), caspofungin (Merck) or tunicamycin (Sigma T7765). TECAN analysis was performed as above. Growth kinetics graphs are an average of 2 biological replicates, with 3 technical replicates each.
To assess the effect of the drugs on filamentation, the wild-type strain was transferred via 96-well replicator (Singer Instruments) from an overnight culture into 100 μL of YEPD with the indicated amount of drugs overnight at 37°C, and then pinned into 100 μL of YEPD with 10% serum in the absence or presence of the drug. Images of the cells were taken after 4 hours at 37°C.

Essentiality was determined as previously described^{7 (link)}. During strain construction, each strain was struck onto YNB-glucose media, and growth in the absence of tetracycline was compared to growth in the presence of 100 μg mL⁻¹ tetracycline after 48 hours at 30 °C. The essential genes were identified using an independent method of streaking strains onto YNB containing 2% glucose, 1000 μg mL⁻¹ 5-flurorotic acid (5-FOA) and 100 μg mL⁻¹ uridine to select for ura- cells which have excised the transactivator, removing transcription of the target gene. Essentiality of each strain in the NRC-distributed GRACE collection was verified by comparing the colony size of each strain transferred via 96-well replicator (Singer Instruments) from overnight cultures to YEPD agar plates in the absence or presence of 1 μg mL⁻¹ DOX and grown at 30°C for 48 hours. Additional analysis comparing colony sizes in the presence or absence of 100 μg mL⁻¹ DOX on YNB agar plates was performed for confirmation when necessary. For S. cerevisiae essentiality, phenotypes were obtained from the Saccharomyces Genome Database (yeastgenome.org). Homologs between S. cerevisiae and C. albicans were identified from the Candida Genome Database (candidagenome.org).

The GRACE library was screened by microscopy. 100 μl YEPD cultures were grown overnight in 96-well plates at 30°C under static conditions. The cells were then transferred via 96-well replicator (Singer Instruments) into either rich medium (YEPD) or rich medium containing 0.05 μg mL⁻¹ doxycycline (YEPD DOX) and incubated at 30°C for 18 hours under static conditions. The strains were then inoculated into media with specific filamentation inducing cues in a 96-well format and incubated; 4 hours at 37°C for serum (YEPD with 10% v/v heat-inactivated newborn calf serum (Gibco #26010-066), 5 hours at 37°C for RPMI medium (Gibco #11875-093), and 6 hours at 30°C for Spider medium (1% mannitol, 1% nutrient broth, 0.2% K₂HPO₄). Images of each well were captured on a Zeiss Axio Observer.Z1 (Carl Zeiss) using 40X magnification. The strains were scored for degree of filamentation on a scale from 0 (unable to filament), 1 (very short or pseudohyphae), 2 (short), or 3 (fully filamentous) (Supplementary Fig. 7). To validate phenotypes, individual strain imaging from liquid media was performed after growth in static conditions in the inducing conditions above using differential interference contrast microscopy on a Zeiss Axio Imager.MI (Carl Zeiss) at 40X magnification.