54-2Y
Candida albicans
Protocols: Cell harvesting Initial Growth: Cells were cultured in YPD at 30 °C for 16.5 hours to an OD600 = ~0.8 before collection by (2 min. at 1000 x g at 4 ◦C) and wash by distilled water. Treatment: cells were resuspended in 15 ml pre-warmed final media (YPD or RPMI) and grown at an appropriate temperature (30 ◦C or 37 ◦C respectively) for 90 minutes. OD600 values were recorded (OD600 = ~1.5) followed by RNA extraction. RNA extraction with modifications: Omega EZNA Yeast RNA Kit (VWR) following the kit protocol with two modifications: (1) 30 °C incubation in SE Buffer/2-mercaptoethanol/lyticase solution time was increased to 90 minutes (2) lysis was performed with bead mill at top speed for 30 minutes at 4 °C. DNase I digestion was included prior to final wash steps. RNA was eluted with 50 µL DEPC water (RNase inactivated) into low binding tubes. In addition to absorbance readings (260/280 ~2), quality was analysed by formaldehyde gel electrophoresis. Illumina