title: GSM3498934: 2_Prime; Candida albicans; RNA-Seq
library_construction_protocol: Candida plaques harvested from mice tongues were treated with 1% Triton X-100 for 10 min at room temperature to lyse murine cells. After incubation, candida cell pellet were re-suspended in 1 ml of TRIzol® Reagent (Life Technologies) for 5 min to further remove traces of  murine cells and centrifuged at 4000rpm for 5 min. The cell pellet was re-suspended in 1ml of TRIzol reagent and vortexed (4 cycles, 6m/s) with 0.45 µm glass beads using a FastPrepTM-24 instrument (MP Biomedicals). Lysed cells (1 ml) were collected and chloroform (200 µl) was added, then mixed vigorously for 15 sec and maintained for 2-3 min at room temperature. The cell lysate was centrifuged at 21000 x g for 10 min at 4°C to separate the RNA containing upper aqueous layer, which was collected and mixed with 0.5 volume of 100% ethanol to precipitate total RNA from C. albicans cells. The total RNA was further purified using RNAeasy minikit from Qiagen according to manufacturer?s instructions. The RNA samples were stored at -80 ºC for the next steps.Total RNA was quantified using Ribogreen Assay (Invitrogen) and the quality of RNA samples were checked using Fragment Analyzer Standard Sensitivity Assay (Advance Analyticals). Illumina TruSeq RNA sample preparation kit (Illumina) was used to prepare cDNA libraries from RNA samples as per manufacturer?s instructions. The cDNA libraries were quantified using Picogreen Assay (Invitrogen) and Library Quantification kit (Kappa Biosciences). Fragment Analyzer High Sensitivity NGS kit (Advance Analyticals) was used to confirm the quality and size of the cDNA libraries. The cDNA libraries were then normalized, multiplexed and sequenced using the Illumina HiSeq2500 following the manufacturer's instructions at the UB Genomics and Bioinformatics Core Facility (Buffalo, NY). Raw RNA-sequencing reads were generating using the illumina HiSeq 2500, using a 50-cycle single read flowcell.