Computational protocol: RNA-Seq Based Transcriptome Analysis of the Type I Interferon Host Response upon Vaccinia Virus Infection of Mouse Cells

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Protocol publication

[…] Libraries were sequenced using TruSeq SBS Kit v3-HS (Illumina) on an Illumina Hiseq 2000 machine at the Max Planck Institute for Molecular Genetics, Berlin. More than 108 100 nt paired-end reads were obtained from each sample and after quality assessment with package FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), the fastq files containing these reads were mapped to the mouse genome (build GRCm38 from Mus musculus C57BL/6J strain) together with the VACV WR genome (Genebank, AY243312.1) using Tophat v2.0.4 with default parameters []. Only those reads aligned against mouse genome were considered in a differential gene expression analysis with Cuffdiff (Cufflinks v2.1.0 software []). Since biological duplicates of samples from untreated cells were available, all comparisons were performed against this sample using the default mode of Cuffdiff, which is the most suitable for our kind of data. Pathway analysis of the significantly differentially expressed genes detected was performed using Ingenuity Pathway Analysis (IPA) software. Creation of proportional Venn diagrams and gene expression heatmaps were generated with the R “VennDiagram v1.6.9” and “Gplots” packages, respectively. The raw RNA-seq data has been deposited at the European Nucleotide Archive (ENA) under the project number PRJEB15047. [...] To evaluate the expression levels of selected genes by RT-PCR, 1 µg of DNA-free total RNA isolated from L929 cells (three biological replicates per condition) was used for first strand cDNA synthesis with iScript cDNA Synthesis (BioRad) using oligo(dT) and random primers. Quantitative polymerase chain reaction (qPCR) analysis was performed using Fast SYBR Green PCR Master Mix (Applied Biosystem) with three technical replicates for each biological replicate, according to the manufacturer's recommendation in an ABI 7900 HT system (Applied Biosystem). Gene-specific qPCR primers were designed using primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) and described in Table S1 in Supplementary Material available online at https://doi.org/10.1155/2017/5157626. Amplification was real-time-monitored and allowed to proceed in the exponential phase, until fluorescent signal reached a significant value (Ct). The fold change was determined using the 2−ΔΔC(t) method []. […]

Pipeline specifications

Software tools FastQC, TopHat, Cufflinks, IPA, gplots, Primer3
Databases ENA
Applications RNA-seq analysis, qPCR
Organisms Vaccinia virus, Mus musculus
Diseases Vaccinia