Analysis of gene expression
Gene expression was quantified using a quantitative PCR method. RNA from frozen cells obtained from the previous step was extracted using a NucleoSpin RNA® (Macherey-Nagel, Germany), purified with rDNase to reduce DNA contamination and quantified using an Agilent 2100 Bioanalyzer (Mx3005P, Stratagene, Germany) in conjugation with the RNA 600 Nano assay. Reverse transcription reactions were conducted to convert the RNAs to cDNAs, which were subsequently quantified using an Agilent Mx3005P qPCR. Primers used for quantification were specifically designed for genes of interest in S. cerevisiae using the NCBI Primer-BLAST Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) with the target PCR product size set at approximately 100 bp, as listed in Table S1. Relative gene expression was calculated using the 2-ΔΔCt method, and the TUB1 gene was used as an internal reference gene.