Illumina NovaSeq 6000 paired end sequencing; RNA-seq of mouse alveolar macrophages from control and Influenza A virus infected animals
RNA-seq of mouse alveolar macrophages from control and Influenza A virus infected animals
Mice were euthanized with CO2. Lungs were removed aseptically, minced, and placed in 1mL of ice cold sterile 2% FCS (in PBS with 2mM EDTA). To maintain sterility and minimize activation of AMs, lungs were gently pressed through 100μm filter using a 3mL syringe without collagenase digestion and washed with excess volume of 2% FCS (in PBS with 2mM EDTA). Cells were spun down at 1500 RPM for 10 minutes at 4°C. Supernatant was discarded. Residual red blood cells were lysed using ACK (Gibco) for 5 minutes. Cells were stained for viability using fixable viability dye (eBioscience), followed by blocking (2.4G2) and then staining for anti-CD45, anti-SiglecF,anti-CD11c for 20 minutes, in dark at 4°C. 200,000-300,000 Viable AMs (CD45+ CD11c+ SiglecF+) were sorted using FACSaria (BD) and immediately resuspended in Trizol (ThermoFisher). RNA was extracted utilizing the Zymo Direct-zol Microkit (Zymo Research) according to the manufacturer's protocol and checked for quantity and quality via spectrophotometry and Agilent bioanalyzer (Agilent technologies) TruSeq Stranded Total RNA
Experimental Factor: infect
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