poly(A)RNASeq of Hepatitis E virus (HEV) transfected Huh7 cells at 72hrs
Huh7 cells were transfected with 4 ľg of in vitro synthesized HEV full length amplicon (FJ457024) using Lipofectamine LTX reagent (Life technologies, United Sates) and cultured for 24hrs and 72hrs post transfection in separate T25 flasks. At the respective time points, total RNA from approximately 1.2 million cells was extracted using Trizol reagent (Life technologies, United Sates) and processed using mirVana miRNA isolation kit (Life technologies, United States) to separate small RNA fraction from total RNA.  After addition of ERCC Exfold spike-in mix (Life technologies, United States), Poly(A) RNA fraction was isolated using Dynabeads mRNA Direct micropurification kit (Life technologies, United States) as per manufacturers instructions. 100ng of poly(A) RNA was then used for preparing sequencing libraries using Ion RNA-Seq kit v2 (Life technologies, United States). Briefly, the poly(A) RNA was fragmented with RNAase, ligated with adaptors and reverse transcribed to cDNA, which was then amplified. Size distribution and concentration of each library was determined on Bioanalyzer 2100 DNA1000 chip (Agilent, United States). Samples were taken in equimolar concentrations and template preparation was done using Ion PI template OT2 kit (Life technologies, United States). Briefly, libraries were bound and amplified on Ion sphere particles in an emulsion PCR (Ion OT2) and enriched on Ion One Touch ES as per manufacturers instructions. Single end, forward sequencing of these templates was then done with Ion PI chip on Ion torrent Proton platform using Ion PI sequencing reagents 200 v3 kit (Life technologies, Unites States).