GSM8282316: PLC124 Fluconazole R3; Candida albicans; RNA-Seq
Total RNA was extracted from frozen cell pellets using the RiboPure-Yeast RNA Isolation Kit (Thermo Fisher) and RNA concentrations were measured using a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Ribosomal RNA was depleted from ~1 µg of total RNA using riboPOOLs (siTOOLs Biotech) kit as recommended by the manufacturer. After depletion samples were precipitated using 2.5 volumes of 95% (vol/vol) ethanol, a 1/10 volume of 3M sodium acetate and 1 µL of glyucoblue, mixed and incubated overnight at -20 °C. Concentration was done by centrifugation at 14000 x g, 30 minutes at 4°C, the pellets were washed twice with 70% ethanol, dried and resuspended. For library preparation, we used the NEBNext Ultra directional RNA Library Prep kit for Illumina (New England BioLabs) as recommended by the manufacturer for relatively low input,13 amplification cycles, and mean library size was around 350bp.