title: pHW-NS1(1–73)Dmd-GFP-NEP C1398T - C1401T sequencing after Covaris fragmentation run 2
design: 500 ng of the pHW-NS1(1–73)Dmd-GFP-NEP C1398T - C1401T plasmid 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 and 2×250 bp MiSeq paired-end sequencing.