Deoxyribonuclease I (DNase I) enzyme (Invitrogen) was used to remove the DNA contamination from RNA samples,if any. Briefly,1 μg of RNA was subjected to DNase I digestion by using 1 U of DNase I in a 10 μl reaction mixture which contained 1X DNase I buffer and appropriate volume of water. The reaction mixture was incubated at room temperature for 15 min. Post incubation,to inhibit DNase I enzyme activity,1 μl of 25 mM EDTA was added to the reaction mixture and tubes were heated at 65°C for 10 min. DNase I-digested RNA samples were used as template to perform PCR for the amplification of CgACT1geneandabsence of amplification product was used as criterion toconfirmproper DNase I digestion and lackof DNAcontaminationin the RNA sample

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation

phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with

Cells were grown in 35 mm dishes at 20% initial confluence. At 40-50% confluence, cells were treated with different genotoxic agents as described in Section 2.2.2. Post treatment, cells were washed twice with PBS and replaced with fresh media to allow recovery for different lengths of time. At each time point, cells were harvested and fixed with 70% ethanol at -20°C overnight. Fixation was carried out by adding 70% ethanol drop by drop, while the cells were being vortexed at a low speed.The fixed samples were brought to room temperature, pelleted down at 2000 rpm for 3 min and washed twice with PBS. Cells were stained with PI solution containing 0.1% Triton X-100, 0.2 mg RNase and 20μg propidium iodide and incubated at 37ºC for 30 min in the dark. Samples were analyzed by flow cytometry (FACS ARIA, BD). Data was analyzed usingFACS DIVA (BD) and FlowJo (FLOWJO, LLC) softwares to identify different stages of the cell cycle