mRNA was purified from total RNA (isolated from control (n = 2) and HEV replicon transfected hepatoma cells at 24hrs (n = 2) and 72hrs (n = 2) post transfection) using Dynabeads mRNA Direct micro purification kit (Cat no: 61021, Life technologies, Carlsbad, California, United States) as per manufacturer’s instructions and quantitated using Qubit (Life technologies, Carlsbad, California, United States). Total RNA samples were spiked with appropriate quantity of ERCC Exfold controls as per given directions (ERCC, Cat no: 4456739 Life technologies, Carlsbad, California, United States) prior to mRNA isolation. 100 ng of ERCC spiked mRNA from each control and test sample was used for library preparation in separate reactions using Ion RNA Seq kit v2 (Cat no: 4475936, Life Technologies) as per manufacturer’s guidelines. Concentration and size distribution of all libraries was determined using DNA1000 chip (Agilent, Santa Clara, California, United States) on Bioanalyser 2100. Clonal amplification of cDNA libraries was performed by using Ion PI template OT2 Reagent 200 v3 (Cat no: 4488318, Life technologies, Carlsbad, California, United States). Template positive ISPs were recovered, enriched and processed for single-end forward sequencing on Ion Proton next generation sequencer using Ion PI chip and Ion PI sequencing reagents 200 v3 (Cat no: 4488315, Life technologies, Carlsbad, California, United States) as per manufacturer’s guidelines. Processing and analysis of RNA Seq data was done as previously described. Briefly, output reads were trimmed, aligned and mapped using Star Aligner and Bowtie2 aligner in Partek flow software and differential expression analysis was done in Partek Genomic Suite version 6.6. Differential expression analysis was performed on RPKM normalised and log transformed read counts by analysis of variance (ANOVA). Genes with fold change > = 2 and p-value < 0.05 were considered for differential expression and gene ontology analysis Full paper
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