Computational protocol: Peromyscus leucopus mouse brain transcriptome response to Powassan virus infection

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Protocol publication

[…] Host mRNA sequencing used the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA), beginning at the poly-A selection step. Libraries were bar coded, amplification cycles were reduced to 10, and libraries were run as a pool across all eight lanes of the HiSeq 2500 sequencer (Illumina, San Diego, CA).The generated RNA-Seq reads for each mouse brain were trimmed of adaptor and poor quality sequences and mapped against the P. maniculatus genome (assembly Pman_1.0) using TopHat 2 software (Kim et al. ). We aligned the RNA-Seq reads to the genome of P. maniculatus because a complete P. leucopus genome is currently not available. We also mapped the RNA-Seq reads to the genomes of the rat (Rattus norvegicus assembly Rnor_6.0), the house mouse (Mus musculus assembly GRCm38.p5), and the Chinese hamster (Cricetulus griseus assembly CriGri_1.0). Using the Basic Logic Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov), we compared selected amino acid sequences of P. leucopus proteins deposited in GenBank to those of P. maniculatus, Rattus norvegicus, Mus musculus, and Cricetulus griseus.Transcript quantification and differential gene expression analysis was performed for each POWV-infected mouse using the Cufflinks software suite (Trapnell et al. ). The triplicate data for each time point were filtered at ± 2-fold change and a false discovery rate (FDR) or corrected p value (q value) of < 0.05. Filtered data were used to generate pathways, networks, and functional inference using Qiagen’s Ingenuity® Pathway Analysis (IPA®) software (Qiagen Redwood City; www.qiagen.com/ingenuity). The sequence data were submitted to the National Center for Biotechnology Information Small Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/), BioProject number PRJNA395043. […]

Pipeline specifications

Software tools TopHat, Cufflinks, IPA
Application RNA-seq analysis
Organisms Powassan virus, Human poliovirus 1 Mahoney, Mus musculus
Diseases Ectromelia, Infectious, Encephalitis, HIV Infections