Computational protocol: Cellular microRNA miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by activating innate antiviral immunity

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Protocol publication

[…] Following transfection with miRNA-26a or miR-Ctr, MARC-145 cells were mock infected or infected with PRRSV VR2332 at MOI = 2 and then harvested 24 hpi. Total cellular RNA was prepared according to the manufacture’s protocol using an RNA Isolation Kit (Invitrogen). All of the RNA Integrity Number (RIN) values were >7.0, and the 28S:18S rRNA ratios were >1.9, as confirmed using an Agilent Bioanalyzer.cDNA libraries were constructed from poly(A)-enriched RNA using Illumina kits and sequenced by 2*100 paired-end sequencing on an Illumina HiSeq 2000 instrument. The FASTQ read files for the four samples were used for further data analysis. Data for gene counts were obtained using the Mayo Clinic pipeline and Burrows-Wheeler Alignment (BWA). To analyze the Illumina reads, TopHat and Cufflinks were used to investigate the DEG profiles and changes in transcript abundance in the miR-26a- or miR-Ctr-transfected MARC-145 cells with or without PRRSV infection. Four files of transcriptome data from the miR-Ctr-, miR-Ctr/V-, miR-26a- and miR-26a/V-inoculated groups were aligned to the UCSC Rhesus Macaque genome build in preparation for differential expression analysis. The four files were processed through Cufflinks to assemble the aligned RNA-seq reads into transcripts and estimate the abundances in FPKM of the paired–end reads. The Cufflinks q-value was the false discovery rate (FDR)-adjusted p-value of the uncorrected test statistic; the q-value used in this study was 0.05. The significance status was “yes” when p > q after Benjamini-Hochberg correction for multiple-testing. Cuffmerge was then used to create a single transcript dataset from the multiple reconstructions. Two runs were then conducted using the miR-26a vs. miR-26a/V and the miR-Ctr vs. miR-Ctr/V datasets and the Cuffdiff program to test for differential expression and regulation among the two disease states. Gene annotation of all significant hits was then performed using a MySQL database matching to the Ensembl Sscrofa 9.56 reference genome currently supported by the Integrative Genomics Viewer (Broad Institute). Finally, heat-maps were analyzed using Cluster and TreeView-1.1.6r2-win software. […]

Pipeline specifications

Software tools BWA, TopHat, Cufflinks, IGV, TreeViewX
Applications Phylogenetics, RNA-seq analysis
Organisms Sus scrofa, Porcine reproductive and respiratory syndrome virus
Diseases Porcine Reproductive and Respiratory Syndrome