RNA-Seq of influenza A/Puerto Rico/8/1934 (H1N1) virus infected monoclonal cell lines with depressed IFITM3 gene activity
RNA-Seq of influenza A/Puerto Rico/8/1934 (H1N1) virus infected monoclonal cell lines with depressed IFITM3 gene activity
In this study influenza A/Puerto Rico/8/1934 was used to infect WI-38 VA-13 cells. Mock-infected cells were included at analysis as controls. Following the incubation period, the cell monolayer was washed twice with PBS and was directly lysed by adding phenol-containing LIRA reagent (Biolabmix, Russia). WI-38 VA-13 cells were cultured in Gibco MEM medium supplemented with GlutaMAX (Thermo Fisher Scientific), 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and sodium pyruvate at 37°C with 5% CO2.  Cells were mock infected or infected with influenza A/Puerto Rico/8/1934 virus (from the collection of Smorodintsev Research Institute of Influenza, Ministry of Health of the Russian Federation) at a dose of 1 TID50/cell and incubated at 37°С for 24 h. Total RNA was extracted from cells using phenol/chloroform extraction with LRU RNA extraction kit (Biolabmix, Russia) according the manufacturer's protocol and analyzed on a 1.5% agarose gel and using an Agilent 2100 Bioanalyzer (Agilent Technologies). RIN > 7 was set as the threshold RIN. The quality of the Input RNA was assessed by the Agilent Bioanalyzer 2100 System and RNA 6000 Nano Chip (Agilent Technologies). For PolyA mRNA enrichment, high quality RNA with a RIN score > 7 was used. ~1 µg total RNA was used for library construction. PolyA mRNA enrichment and construction of RNA-seq libraries were performed with the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB) correspondingly. Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system using Qubit DNA HS Assay Kit (Thermo Fisher Scientific). The quality control analysis of library was assessed using the Agilent Bioanalyzer 2100 System and DNA High Sensitivity Chip (Agilent Technologies).