# Longer Ubiquinone Side Chains Contribute to Enhanced Farnesol Resistance in Yeasts
## 2 Materials and Method
### Ulbiquinone Analysis in C. albicans  
To analyze ubiquinone content in _C. albicans_, the cells were grown in 40 mL of either rich (YPD) or minimal media (GSB, GPP with N-acetylglucosamine, or SC) at 30 or 37 \({}^{\circ}\)C for 18-24 h as appropriate to induce the different morphological stages of _C. albicans_, i.e., yeasts or mycelia, white or opaque cells, or anaerobically grown cells. The morphology of each cell type was confirmed by microscopy before harvesting. Cells were harvested by centrifugation at mid-exponential phase, washed once with 25 mL of water, and resuspended in 1 mL of water whereupon cell number was quantified by absorbance at 600 nm and the cell numbers normalized by adjusting the volume of each sample. One ml of each suspension was transferred to a 10 mL pyrex tube containing 0.5 mL of 0.5 mm glass beads, spiked with 8.5 nmoles of UQ-10, and vortexed for 90 s. Samples were then mixed with 2 mL of 95% ethanol, heated at 70 \({}^{\circ}\)C for 10 min, cooled and extracted twice with 5 mL hexane. The hexane layers were combined, evaporated to dryness with N\({}_{2}\), and resuspended in 1 mL of methanol: dichloromethane (10:1). The types of UQ present in the ubiquinone pool were identified as described by [PERSON] et al. [21] using high-performance liquid chromatography (HPLC) in diode array detection (DAD) mode with UQ10 as the internal standard [21]. Extracts from cell suspensions were analyzed at 30 \({}^{\circ}\)C by HPLC (Agilent, Santa Clara, CA, USA) on a 5 \(\upmu\)M Supelco Discovery C-18 column (250 \(\times\) 4.6 mm, Sigma-Aldrich, St. Louis, MO, USA) in isocratic mode at a flow rate of 1 mL min\({}^{-1}\) with methanol:hexane (95:5 \(v\)/\(v\)) for spectrophotometric detection of quinones at 275 nm. Quinol species had been fully reoxidized during heating and were quantified as a part of the quinone pool. Retention times for the ubiquinones were 8.6 min for UQ-6, 11.5 min for UQ-7, 15.8 min for UQ-8, 22.1 min for UQ-9, and 31.5 min for UQ-10. The typical recovery value for yeast ubiquinones is approximately 90% and data were corrected accordingly. All glassware used for culture growth and analysis were washed with 2M NaOH and then rinsed with Milli-Q water followed by a final rinse in acetone.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Yeast Strains** & **Genotype Description** & **Source** & **UQ** \\ \hline \multicolumn{4}{c}{_C. albicans_ strains} \\ \hline SC5314 & Wild type (clinical isolate) & [29] & UQ9 \\ \hline WO-1 & MTL\(a\) frequent white/opaque switching & [30] & UQ9 \\ \hline \multicolumn{4}{c}{_S. cerevisiae_ strains} \\ \hline BY4741 parent & BY4741 _MATa his3\(\Delta\)I leu2\(\Delta\)0 met15\(\Delta\)0 \(ura3\Delta\)0 rho\({}^{+}\)_ & [21] & UQ6 \\ \hline \(\Delta\)\(coq1\):pYES & _coq1_ knockout transformed with empty vector & [21] & none \\ \hline \(\Delta\)\(coq1\):pYES + _At2g34630_ & _coq1_ knockout transformed with Arabidopsis _At2g34630_ & [21] & UQ9 \\ \hline \(\Delta\)\(coq1\):pYES + _COQ1_ & _coq1_ knockout transformed native _COQ1_ gene & [21] & UQ6 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Strains used in this study.