title: Infected_WT_Day6_3
organism: Mus musculus
description: Protocols: Mice were housed in an enhanced barrier specific pathogen-free facility under a strict 12-hour light cycle and fed irradiated chow diet (PicoLab Rodent Chow 20, Purina Mills). Mice were maintained on a C57BL/6 background. A heterozygous x heterozygous breeding scheme was used to generate Irgm1-/- and wildtype littermate control mice. All study protocols were approved by the Washington University Animal Studies Committee. Recombinant influenza A/WSN/33 (rWSN) H1N1 virus strain was used. For influenza infections, 8-12 week-old male and female mice were anesthetized using isofluorane and injected intranasally with 5,000 pfu virus in 40 µl total volume of PBS (20 µl per nare). Uninfected mice were sacrificed on Day 0. Infected mice were sacrificed 3, 6 or 10 days post-infection and the right caudal lung lobes were harvested for RNA. Total RNA was isolated from whole lung homogenates with Trizol Reagent according to the manufacturer's protocol. RNA was DNase-treated and purified via Qiagen RNA Purification columns according to the manufacturer's instructions. Total RNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm. Total RNA quality was then determined by Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturer's recommendations. Library preparation was performed by the Genome Technology Access Center (GTAC) at Washington University. Library preparation was performed with 1ug of total RNA. Total RNA integrity was determined using an Agilent bioanalyzer. Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina-EpiCentre). mRNA was then fragmented in buffer containing 40mM Tris Acetate pH 8.2, 100mM Potassium Acetate and 30mM Magnesium Acetate and heating to 94 degrees for 150 seconds. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-2500 using single reads extending 50 bases.