title: RNA-Seq of Huh7: IgG-IP control2
design: Total RNA was isolated from Huh7 cells treated with IFNbeta or vehicle  as well as from anti-MDA5- and IgG-RNA-IP samples by TRIzol reagent (Invitrogen) following the manufacturers instructions. RNA libraries were prepared using SMART-Seq Stranded kit (Takara Bio Inc., USA) following the manufacturer's recommendations. In brief, RNA samples were fragmented and reverse transcribed into first strand cDNA using random hexamers. Double-stranded cDNAs were end-repaired and adenylated (singly) at the 3' ends. Barcoded adapters containing unique six-base index sequences and T-overhangs were ligated to the cDNA samples prepared from each of the samples. Individual cDNA library was subjected to ribosomal cDNA depletion, amplified with PCR and purified with AMPure beads. The barcoded libraries were pooled in equimolar (10nmol/L) amounts and diluted to 4 pmol/L for cluster formation of a single flow cell lane, followed by pair-end sequencing on an Illumina NovaSeq 6000 sequencer. After demultiplexing the sequencing data, adapter sequences were removed and the individual libraries were converted to the FASTQ format. Sequence reads were mapped to the human genome (hg38) with Hisat2, allowing up to two mismatches. Sequence reads aligned to the human genome were imported into Partek Genomics Suite version 6.6 (Partek, St Louis, MO) for sequence read clustering, counting and annotation. The RefSeq transcript and LNCipedia version 5.2 databases were chosen as the annotation references for mRNA and lncRNA analyses, respectively. The read counts of each known transcript were normalized to the length of the individual transcript and to the total mapped read counts in each sample and expressed as FPKM (fragments per kilobase of exon per million mapped reads). Sequence reads mapped to different isoforms of individual genes were pooled together for subsequent comparative analysis. Differential expression, heat-map and hierarchical clustering analyses of mRNAs and lncRNAs between groups were performed using Partek Genomics Suite 6.6, where normalized read counts, fold change, and P values of each transcript were reported. A false discovery rate (FDR)-controlling procedure was used to correct for multiple comparison. An FDR-adjusted p-value <0.05 was considered significant. Gene ontology (GO) and pathway analyses of the transcripts that were differentially expressed in IFN-treated Huh7 cells were performed using WebGestalt (WEB-based Gene SeT AnaLysis Toolkit).