GSM3702158: YC1_PMT12-BF4; Candida albicans; RNA-Seq
Cells were centrifuged at 4°C for 10 min at 3,250 rpm, and washed with ice-cold ddH2O to discard medium. Total RNA was extracted using TRIzol reagent (Invitrogen, USA). Collected cells were freeze in liquid N2, and vortexed with beads. After adding 1 mL TRIzol, cultures were centrifuged at 4°C /12,000g for 10 min, and the supernatant was transferred to a new tube. After 5 min incubation at room temperature, 200 μL of chloroform was added and mixed thoroughly, and tubes were incubated at room temperature for 3 min, centrifuged at 4°C/12,000g for 15 min, and supernatant was transferred to a new tube. Tubes were added 500 μL of isopropanol, incubated at room temperature for 10 min, centrifuged at 4°C/12,000g for 10 min, and the supernatant was discarded. Pellet containing RNA was washed twice with 75% ethanol and resuspend with RNase free water. The mRNA was enriched with oligo(dT) magnetic beads and shortened into approximately 200-base fragments in fragmentation buffer. The first strand of cDNA was synthesized by the use of a random hexamer, buffer, dNTPs, and RNase H, and the second by the use of DNA polymerase I. The double strand cDNAs were purified with magnetic beads. After end preparation and 3' end single nucleotide adenine addition were performed, sequencing adaptors were ligated to the fragments, and amplified by PCR. An Aglient 2100 bioanalyzer and ABI StepOnePlus real-time PCR system were used to qualify and quantify the sample library, and the library products were sequenced via an illumina HiSeq 2000 instrument.