title: RNAseq of influenza A virus (A/WSN/1933): wild-type replicate 3 passage 7
design: Viral RNA collected from cell culture supernatants and reverse-transcribed with Tuni-12 primer (5- ACGCGTGATCAGCAAAAGCAGG). PCR was done with Tuni-13 (5- ACGCGTGATCAGTAGAAACAAGG) and Tuni-12 primers for 30 cycles. The PCR products were purified using the Monarch PCR and DNA Cleanup Kit (NEB), and the DNA concentration and purity were assessed using Qubit dsDNA broad range kit (Invitrogen). The pooled amplicons from each sample were first cleaned up with 0.56x Ampure XP beads to remove short DNA fragments, and then sheared on Covaris LE220 sonicator to around 300 bp. Illumina sequencing adapters and barcodes were added to the sheared DNA fragments using a NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, MA) following the guide from the manufacturer. The purified DNA library products were examined on Bioanalyzer DNA High Sensitivity chips (Agilent, CA) for size distribution, quantified by Qubit fluorometer (Invitrogen, CA), and pooled at equal molar amount. The library pool was sequenced on Illumina MiSeq using a Nano 300 cycle kit as pair-end 150 nt reads. Raw sequencing reads were filtered by the MiSeq control software and only the Pass-Filter (PF) reads were used for further analysis.