title: RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) Replicon Transfected Huh-7 cells-replicate 1.
design: mRNA purification    mRNA was purified from total RNA using Dynabeads mRNA direct micro kit (Life technologies, USA) as per manufacturer’s directions. Before the start of the protocol, each sample was spiked with ERCC spike-in controls (Life technologies, USA) proportionate to the quantity of total RNA in each sample. Purified mRNA was quantified in Qubit and the integrity of the mRNA was checked using a Bioanalyzer 2100 (Agilent, USA) using RNA6000 pico chip and kit as per manufacturer’s instructions.    Library preparation  Library preparation for RNA-Seq involved four steps including fragmentation, adaptor ligation, cDNA synthesis, and cDNA amplification. All the steps were achieved using Ion-PGM RNA-Seq kit v2 (Life technologies, USA) as per manufacturer’s instructions. Equal quantity of mRNA (150 ng) was used as input from all the samples. The amplified cDNA was quantified in Qubit, and its profile checked for size distribution and peak concentration on Bioanalyzer 2100.      Template preparation and sequencing    Amplified cDNA fragment libraries were further clonally amplified by emulsion PCR on Ion Sphere Particles (ISP’s) using Ion One touch 200-template kit v2 DL (Life technologies, USA) in Ion One Touch v1 (Life technologies, USA) as per manufacturer’s instructions. Post PCR the template positive ISP’s were recovered and were enriched to remove non-template ISP’s on Ion OneTouch ES (Life technologies, USA) as per manufacturer’s protocol. The enriched ISP’s were recovered and processed for sequencing using Ion PGM 200 sequencing kit as per manufacturer’s instructions. Finally the ISP’s were loaded onto a 318 chip and sequenced on Ion torrent-PGM (Life technologies, USA) using default parameters (single-end, forward sequencing).