title: Pacbio SEQ2 Short insert library: Glioblastoma-2
design: Two glioblastoma tissues were obtained from surgical resection of lesions from patients with pathological diagnosis from the Mount Sinai Hospital. Informed consent from both patients was obtained in accordance with an IRB-approved protocol (HS# 10-00135). Glioblastoma-1 was derived from the anterior hippocampal lesion of an 85-year old female patient and glioblastoma-2 was derived from the nodular inferolateral component of a 58-year old male patient. Genomic DNA from both tissues was extracted using a DNeasy Kit (Qiagen) following manufacturers instructions. The short-insert DNA SMRTbell library was performed according to the manufacturers instructions by first shearing the sample to ~200bp using a Covaris LE220 Focused-ultrasonicator,followed by damage and end repair and ligation of single-stranded barcoded adaptors. Sequencing primer was then annealed to the SMRTbells and incubated with the template for 1 hour at 20 C using a ratio of 20:1. The Sequel 2.1 polymerase was then bound to the annealed template complex using a ratio of 30:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 C. The binding complex was cleaned up, diluted to the final loading concentration, and held at 4 C until diffusion-based loading on the Sequel II instrument. Diluted binding complex at concentration of 125-175pM was loaded on the 8M sequencing chip and 30 hour movies were recorded according to the manufacturers guidelines.