title: Small_A549_0h_2
organism: Homo sapiens
description: Protocols: A549 cells were maintained in Gibco DMEM/F12 medium supplemented with GlutaMAX and 10% FBS 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 or 48 h. In this study influenza A/Puerto Rico/8/1934 was used to infect A549 cells. Mock-infected cells were included at analysis as control. Following the incubation period, the cell monolayer was washed twice with PBS and was directly lysed by adding phenol-containing LIRA reagent (Biolabmix, Russia). Small RNA (<200 nucleotide length) 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% TAE-agarose gel and using Bioanalyzer 2100 instrument (Agilent, USA) with an Agilent Small RNA kit. The quality of the Input RNA was assessed by the Agilent Bioanalyzer 2100 System and Agilent Small RNA kit (Agilent, USA). ~0.5 µg of enriched small RNA (<200 nucleotide length) was used for library construction. The construction of cDNA libraries according to a standard protocol using a NEBNext Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs, UK), as well as massive parallel sequencing on a NextSeq Illumina 500 platform, were conducted at the Institute of Fundamental Medicine and Biology, Kazan Federal University (Kazan, Russia). Libraries 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 libraries was assessed using the Agilent Bioanalyzer 2100 System and DNA High Sensitivity Chip (Agilent, USA).