Transcriptome analysis of Candida auris treated with tunicamycin
Transcriptome analysis of Candida auris treated with tunicamycin
Cells were grown in YPD broth at 30˚C from OD600=0.01 till OD600=1.0.The culture was divided to two group: One group was treated with 4 μg/mL tunicamycin. The other group was treated with equal volume of DMSO. 3 hours later, pellets of 5ml cell culture were obtained by centrifuging at maximum speed for 1min. the pellets were fast frozen in liquid nitrogen.  Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was measured using a NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA). The RNA concentration was measured using the Qubit® RNA Assay Kit was used in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed with the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA), following the manufacturer's protocol. Index codes were added to attribute sequences to each sample. mRNA was purified from the total RNA with poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under an elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). A random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-) were used to synthesize the first strand cDNA. Then, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Exonuclease/polymerase activities were used to convert the remaining overhangs into blunt ends. After adenylation of the 3' ends of the DNA fragments, the NEBNext Adaptor with hairpin loop structures was ligated to prepare for hybridization. To select cDNA fragments of 150–200 bp in length, the AMPure XP system (Beckman Coulter, Beverly, USA) was used. 3 μl of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by a 5 min incubation at 95°C before PCR. PCR was then performed with the Phusion High-Fidelity DNA polymerase, Universal PCR primers and the Index (X) Primer. Finally, the PCR products were purified (AMPure XP System), and the library quality was assessed using the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed using the cBot Cluster Generation System and the TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer's instructions. 