Computational protocol: Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects

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Protocol publication

[…] For analyses of the RNASeq data, all treatments were normalized to their respective control and gene names were converted to human HGNC gene names before bioinformatical analyses were performed. Ingenuity Pathway Analysis (IPA; Ingenuity Systems, http://www.ingenuity.com/) was used to analyze activated pathways and predicted upstream activators as previously described [, ]. In brief, activation of Z values were used to determine activation of pathway or regulators (−2 ≥ Z, significant inhibition; 2 ≤ Z, significant activation), p values were calculated using right-tailed Fisher’s exact representing the significance for the overlap between dataset and pathway or regulator. Gene set enrichment analysis (GSEA; http://www.broadinstitute.org/gsea) was performed to evaluate functional gene sets that are associated with each of the treatments compared to controls. The fold-change regulation of the genes after treatment was used to perform pre-ranked GSEA. The BIOCARTA, KEGG, and REACTOME databases were queried; p values were estimated by 100 permutations and a null-distribution of the enrichment scores. P values were adjusted using the Benjamini-Hochberg method. A normalized enrichment score (NES) was computed by mean normalization []. The Connectivity Map database (http://www.lincscloud.org/; []) was used to analyze upstream regulators based on regulated genes after IFNαB/D or supernatant treatment. The top 50 expressed genes and top 50 downregulated genes were selected from, each treatment, per analysis, and a connectivity score >90 or <−90 was considered significant. [...] Data from quantitative reverse transcription PCR (qPCR), bioassay, focus forming assays, and viral spread assays were analyzed with unpaired t test using GraphPad Prism software. Statistical analyses of RNASeq data were performed by GATC Biotech using Cufflinks [, ]. […]

Pipeline specifications

Software tools IPA, GSEA, Cufflinks
Databases KEGG BioCarta
Application RNA-seq analysis
Organisms Human poliovirus 1 Mahoney, Japanese encephalitis virus, Mus musculus, Tick-borne encephalitis virus, West Nile virus
Diseases Encephalitis, Encephalitis, Tick-Borne, Meningitis, Microcephaly, HIV Infections, Tick-Borne Diseases, Flavivirus Infections