Dnase enzyme

Gene expression analysis was performed according to previously described method (Demuyser et al., 2017 (link)). The cells were grown to mid-exponential phase in SC medium supplemented with the carbon source of interest. Cells were washed with ice-cold Milli-Q water, frozen in liquid nitrogen and kept at −80°C. The cell pellet was dissolved in TRIzol (Thermo Fisher), and cells were lysed by fast prepping with glass beads. Afterward, RNA was isolated using chloroform, isopropanol, and 70% ethanol. The RNA was treated with DNase enzyme (New England Biolabs), to remove present DNA fractions, and converted into cDNA by using the iScript cDNA synthesis kit (Bio-Rad). To perform the quantitative PCR, a Go-Taq polymerase (Promega) and a StepOnePlus machine (Thermo Fisher) were used. The results were analyzed by making use of the qBasePlus software (Biogazelle).

S. cerevisiae strains were grown in SDglu medium at 30°C for 24 h, with or without 20 μg/ml fluconazole. C. glabrata and C. albicans cells were incubated for 8 h at 37°C or 30°C in RPMI medium, with or without 1 μg/ml fluconazole. Cells were washed with ice-cold water, resuspended in TRIzol (Thermo, Fisher), and broken using glass beads and a FastPrep machine (MP Biomedicals). RNA was extracted by respective addition of chloroform and isopropanol and washed three times with 70% ethanol. Equal amounts of RNA were treated with a DNase enzyme (New England Biolabs) and converted to cDNA (iScript cDNA synthesis kit; Bio-Rad). Real-time quantitative PCR (qPCR) reactions were conducted using GoTaq polymerase (Promega) and a StepOnePlus real-time PCR device (Thermo, Fisher). Data were analyzed using qBasePlus software (Biogazelle) (79 (link)). Further data analysis and statistics analysis of log₂(Y) transformed expression values were performed with Graphpad Prism. Transformation of the data points was performed to enable the use of standard statistical methods. Graphs show the means of the transformed values, together with their SEM. The statistical method used is mentioned under each figure. Copy number analysis of the genomic DNA of transformants was performed by qPCR, as described above.

Cells were collected after 90 min of adhesion in a six-well tissue culture to obtain the necessary number of cells. The wells were washed with 1× PBS and scraped off the bottom of the wells by making use of a sterile cell scraper. RNA was purified with the RiboPure-Yeast kit from Ambion. The RNA was treated with DNase enzyme (New England Biolabs), to remove present DNA fractions, and converted into cDNA by using the iScript cDNA synthesis kit (Bio-Rad). To perform the quantitative PCR, a Go-Taq polymerase (Promega) and a StepOnePlus machine (Thermo Fisher) were used. The results were analyzed by making use of the qBasePlus software (Biogazelle).