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<p>RNA Isolation and Sequencing Mice were sacrificed 3 days post-infection (dpi) and both lungs were extracted and transferred immediately to RNAlater (Qiagen), stored at 4°C for one day, and then stored at −20°C. RNA was isolated using Qiagen Midi kit (30). RNA quality was evaluated on a 2100 Bioanalyzer (Agilent). Five-hundred nanograms of total RNA was used to prepare libraries for sequencing using the Lexogen SENSE RNA-seq library kit for Ion Torrent. Libraries were amplified for 11 cycles as the final step of library preparation. Before sequencing, 1-μl aliquots were pooled and sequenced on an Ion Torrent PGM 314 chip. Barcoded data from the PGM was used to balance the final pool before sequencing. Library pools were sized to ~260 bp on a Pippin Prep instrument using 2% Pippin agarose gel. The sized libraries were evaluated on an Agilent High Sensitivity chip, quantified using real-time PCR, and used to prepare beads using a One-Touch 2 device. Beads were sequenced on an Ion Torrent Proton P1 chip. On average, 67 million reads were obtained per strain.</p>
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