A/quail/Hong Kong/G1/1997 (H9N2) passage 4 apical and cardiac lobe
150 ng of each purified RT-PCR sample was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 400 bp. The fragment ends of 50 ng of these DNA fragments were repaired using the NEBNext Ultra DNA Library Preparation kit (New England Biolabs), followed by adding the adaptors by using the NEBNext Multiplex Oligos for Illumina kit (Dual Index Primers Set 1, New England Biolabs). The resulting fragments were size-selected for an insert size of 300 to 400 bp using Agencourt AMPure XP bead sizing (Beckman Coulter). Afterwards, indexes were added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt AMpure XP beads. Fragments were analyzed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. After the 2×250 bp MiSeq paired-end sequencing run, the data were base called, generating Illumina FASTQ files (Phred +64 encoding).