RNA sequencing of Cryptococcus neoformans H99 treated with eltrombopag

The gene expression of C. neoformans H99 treated with eltrombopag C. neoformans H99 cells were grown overnight in liquid YPD at 30°C, washed twice with ddH2O, diluted 1:100 in liquid YPD and grown at 30°C with agitation for 3 h to reach a density of 3 × 10^7 CFU/mL. At this point cultures were equally divided into two aliquots to which either 0.06 μg/mL eltrombopag or same amount of DMSO was added, followed by incubation at 30°C for 90 min. After this treatment, cultures were centrifuged and total RNA was extracted using Trizol reagent and treated with DNase I (Ambion, Thermo Fisher Scientific) to degrade contaminating DNA. mRNAs were enriched with oligo (dT) magnetic beads. Fragmentation buffer allowed the mRNA to be shortened into ~200-base fragments. Then, the first strand of cDNA was synthesized using random hexamer, buffer, dNTPs, and RNase H, and the second strand of cDNA was synthesized by DNA polymerase I. The double-stranded cDNAs were purified with magnetic beads. End preparation and 3' end single nucleotide adenine addition was performed, and sequencing adaptors were ligated to the fragments, which were enriched by PCR amplification. During the quality control step, the Agilent 2100 Bioanaylzer and ABI StepOnePlus RealTime PCR system were used to qualify and quantify the sample library. The library products were sequenced via Illumina NextSeq 500.