miRNASeq of Hepatitis E virus (HEV) transfected Huh7 cells at 24hrs replicate
Huh7 cells were transfected with 4 ľg of 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 appx.1.2 million cells was extracted using Trizol reagent (Life technologies, United Sates) and processed using mirVana miRNA isolation kit (Life technologies, Unites States) to isolate small RNA fraction. 100ng of small RNA was then used for preparing sequencing libraries using Ion RNA Seq kit v2 (Life technologies, United Sates). Briefly, small RNA was ligated with adaptors and reverse transcribed to cDNA, which was then amplified using barcoded primers (Ion Express Barcode Adapters 1-16, Life technologies, United Sates). Size distribution and concentration of the libraries was determined on Bioanalyzer 2100 DNA 1000 chip (Agilent, United States). Samples of each time point were pooled together in equimolar concentrations and template preparation done using Ion PI template OT2 kit (Life technologies, United Sates). 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, United Sates).