Lactate- and hypoxia-induced beta-glucan masking in responding and non-responding Candida albicans clinical isolates
Lactate- and hypoxia-induced beta-glucan masking in responding and non-responding Candida albicans clinical isolates
Candida albicans cells were harvested by centrifugation (5,000 rpm, room temp) and the cell pellets flash frozen in liquid nitrogen. Candida albicans cells were incubated overnight in 5 ml volumes in minimal GYNB medium, and then transferred to fresh GYNB medium in 50 mL culture flasks for 5 hours: (a) 1% lactate; (b) 0% lactate control; (c) hypoxia; or (d) normoxic control (30C, 200 rpm). RNA was prepared from frozen cell pellets via Qiazol/chloroform extraction (QIAgen) according to the manufacturerâ€™s instructions and assessed for quality by nanodrop. RNA was DNAse treated (TURBO DNAse, Ambion) according to the manufacturers instructions and assessed for quality by Agilent Bioanalyser (RIN>8). ERCC spike in controls were added to RNA samples to be used as internal controls. Libraries were prepared with the Illumina TruSeq Stranded mRNA kit following manufacturer's instructions.