PR8-NS1(1–73)GFP virus sequenced after Covaris shearing
500 ng of the PR8-NS1(1–73)GFP virus RT-PCR product was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 300-400 bp. Next, the NEBNext Ultra DNA Library Preparation kit (New England Biolabs) was used to repair the ends and to add Illumina MiSeq-compatible barcode adapters to 100 ng of fragmented DNA. The resulting fragments were size-selected 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. The multiplex sample was heat denatured for 2 min at 96°C before loading on the MiSeq chip. After the 2×250 bp MiSeq paired-end sequencing run, the data were base called and reads with the same barcode were collected and assigned to a sample on the instrument, which generated Illumina FASTQ files (Phred +64 encoding).
DMPR - VIB - UGent
PR8-NS1(1–73)GFP virus sequenced after Covaris shearing