GSM4041340: R1_T36_2; Candida albicans; RNA-Seq
5μg total RNA was used for mRNA isolation using Dynabeads OligodT (Invitrogen, Carlsbad, CA), then about 30-50ng isolated mRNA was used for mRNA-Sequencing library preparation by following the BIOO Scientific NEXTflex Directional RNA-seq kit (dUTP-based) sample preparation guide. First, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic Dynabeads. Then, the mRNA was fragmented into small pieces using divalent cautions under elevated temperature (95 °C for 10 mins). The cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H and an end repair process consisting on the addition of a single 'A' base, which is then followed by ligation of the adapters. The products were purified and enriched by PCR to create the final RNA sequencing library. Directionality was retained by adding dUTP during the second strand synthesis step and subsequent cleavage of the uridine containing strand using Uracil DNA Glycosylase. The RNA sequencing libraries were then subjected to quantification process, pooled for cBot amplification and subsequent 100bp paired end sequencing run with Illumina HiSeq 2000 platform. After the sequencing run, demultiplexing with CASAVA was employed to generate the fastq file for each sample. RNA libraries were prepared for sequencing using standard Illumina protocols