RNA-seq of sus scrofa: piglet female lung
Porcine alveolar macrophages (PAMs) were seeded in 60 mm plates and cultured 12 h. Then PAMs were inoculated with PRRSV strain BJ4 at a multiplicity of infection (MOI) of 1 or mock control for 24 h. Total RNA was extracted from cells using TRIzol (Life Technologies) and digestion with DNaseI (TaKaRa, Japan) following the manufacturers instructions. Sequencing libraries were generated using the KC-DigitalTM small RNA Library Prep Kit for Illumina (Catalog NO. DR08602, Wuhan Seqhealth Co., Ltd. China). Sequencing was performed using HiSeq X-10 platform (llumina) with PE50 mode 1. Clean reads were obtained from the raw data reads by fastx toolkit (version: 0.0.13.2) for discarding the low-quality and adaptor sequences were trimmed by cutadapt (version: 1.15) and further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. Clean reads were searched against miRbase to identify know miRNAs in sus scrofa. miREvo and miRDeep software were used to comprehensively predict novel miRNAs based on the hairpin structure or step-loops. The miRNA differential expressed between mock and PRRSV infection groups was identified using the edgeR package (version: 3.12.1). These miRNAs with more than 1.5 times up-regulated expression and more than 50% down-regulated expression were screened. A cutoff of P-value <0.05 and |Log2Fold-change|>5 was used to judge the statistical significance of miRNA expression differences.