RNA-seq of Candida albicans SC5314 grown on minimal media supplemented with glucose or inulin preparations
RNA-seq of Candida albicans SC5314 grown on minimal media supplemented with glucose or inulin preparations
SC5314 C. albicans liquid cultures were grown in a shaking incubator at 37 oC, 200 rpm in YNB (Yeast Nitrogen Base without amino acids) supplemented with 2% glucose or 2% inulin overnight  Fresh media was then inoculated at an OD600 of 0.1 and grown for 3 hrs at 37oC with 200RPM shaking. Cells were subsequently collected via centrifugation for RNA extraction The Ribopure RNA Purification Kit, Yeast (Invitrogen) was used to isolate RNA from C. albicans according to the manufacturer's instructions. Briefly, approximately 3x108 freshly grown C. albicans cells were centrifuged and the supernatant was removed. The lysis buffer, SDS and phenol:chloroform:isoamyl alcohol from the kit were added to the pellet and mixed well. The mixture was added to a screwcap Nunc tube containing zirconia beads and the C. albicans cells sheared for 10 minutes at maximum speed using a bead beater (MP FastPrep 24). After centrifugation for 5 minutes, the supernatant was collected and added to a fresh tube. Binding buffer was added to each sample, followed by 100% ethanol. The mixture was added to a filter cartridge assembled in a collection tube and centrifuged for 1 minute. The supernatant was discarded and the filter cartridge was washed once with Wash Solution 1, and twice with Wash Solution 2/3. In each case, the flow-through was discarded. The filter cartridgewas dried for 1 minute by centrifugation and then the RNA was eluted in preheated elution solution. To remove DNA, DNase I buffer was added to the RNA sample together with DNase I, and incubated at 37°C for 30 minutes. The DNase I was then inactivated via the addition of a DNase Inactivation Reagent for 5 minutes. Finally, the sample was centrifuged to pellet the inactivation reagent, the RNA supernatant was transferred to a fresh tube, and the sample stored at -80 The library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit with poly(A) enrichment, following the manufacturer's standard protocol. Libraries were quantified and quality-checked prior to sequencing.