title: RNA-Seq of Rhodotorula glutinis with irradiation
design: About 10 mg cell pellets samples were used to total RNA extraction by the previous method (Huang et al. 2013) and the purity and concentration of RNA were determined by the NanoPhotometer spectrophotometer (IMPLEN, USA) and the Qubit RNA Assay Kit (Life Technologies, USA), respectively. RNA integrity was assessed using the RNA Nano 6000 Assay Kit (Agilent Technologies, USA). 1.5 g RNA per sample was used to generate sequencing libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEB Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150-200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, USA). Then 3 l USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37C for 15 min followed by 5 min at 95C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturers instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and reads with low quality from raw data.