title: Amplicon sequencing of Influenza A Virus: infected mouse lungs
design: Viral RNA was extracted from 50 L of whole lung homogenate on a Kingfisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) by using the Ambion MagMAX-96 AI/ND Viral RNA Isolation kit (Applied Biosystems, cat# AM1834). RNA concentration was measured spectrophotometrically (Nanodrop). Multi-segment polymerase chain reaction (MS RT-PCR) was performed using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerases (ThermoFisher, cat#12574-035) and influenza-specific universal set of primers (45) (Opti-F1 5-GTTACGCGCCAGCAAAAGCAGG-3, Opti-F2 5-GTTACGCGCCAGCGAAAGCAGG-3, Opti-R1 5-GTTACGCGCCAGTAGAAACAAGG-3). RNA (5 L) was added and placed into a thermocycler paused at 55C. The following cycling parameters were then followed: 1 cycle of 55C/2min; 1 cycle of 42C/60min; 94C/2min; 5 cycles of 94C/30s, 44C/30s, 68C/3.5min; 26 cycles of 94C/30s, 57C/30s, 68C/3.5min; 1 cycle of 68C/10min; and then held at 4C. 5 L of the reaction was analyze by 0.8% agarose gel electrophoresis to verify all genomic segments are present, with PB1 and PB2 migrating together at 2.3Kb and minimal non-specific amplification below 800bp.  The MS RT-PCR reaction was purified using Agencourt AMPure XP Kit (Beckman Coulter) according to manufacturers instructions. Briefly, 40 L of the DNA amplicons were transferred to a 96-well microplate (Biorad) and mixed with Agencourt AMPure XP beads (Beckman Coulter) for magnetic separation of the amplicons. The bound beads were washed twice with 100% ethanol and the purified amplicons suspended in 1x Tris buffer (TE). The concentration of the purified DNA was measured spectrophotometrically prior to storage at -20C (Nanodrop). DNA amplicons were deep sequenced using Illumina MiSeq technology. Deep sequencing was performed by the St. Jude Childrens Research Hospital Hartwell Center with DNA libraries prepared using Nextera XT DNA-Seq library prep kits (Illumina, cat# FC-131-1024) with 96 dual-index bar codes and sequenced on an Illumina MiSeq personal genome sequencer.
library_name: RRH-RWTp5_1