Miseq reagent nano v2

For copy number variation analysis (CNV), copy number abnormalities were assessed in the three Schwann cell lines using a high resolution Cytoscan HD single-nucleotide polymorphism array (Affymetrix, santa Clara, CA, USA). The hybridization, amplification and labeling protocols were performed according to the manufacturer’s recommendations (Affymetrix).
For Next Generation Sequencing (NGS), the Illumina MiSeq technology was used for NF1 mutation screening, alongside the AmpliSeq for Illumina Library Prep (Illumina) for library construction. Paired-end sequencing was performed on the Illumina MiSeq (Illumina) using 300 cycle (Miseq Reagent Nano v2) kit format. Raw signal data were analyzed using in-house bioinformatics pipeline and visualized with the Integrative Genomics Viewer (IGV).

Purified DNA was quantified using the Quant-iT PicoGreen double-stranded DNA (dsDNA) kit (Invitrogen, Australia), with fluorescence measured on a Victor X2 plate reader (PerkinElmer, Australia). Dual-indexed sequencing libraries were prepared using DNA Prep v2 (Illumina, Australia). The size distribution of the library was assessed on LabChip GX Touch24, using the HTDNA high-sensitivity assay (PerkinElmer). Samples were pooled in equimolar quantities and spiked with 10% PhiX. Sequencing was performed using MiSeq reagent Nano v2 on a MiSeq sequencer (Illumina).