Human mixed genomic dna

PCR and Sanger sequencing confirmed the presence of fusion candidates generated by Trellis. Primers were designed 200 bp on either side of the junction (Table S1m). Primerswere purchased from IDT (Coralville, IA) and purified by desalting. Primers and probes were resuspended to 100 μΜ in IDTE (10 mM Tris, pH 8.0; 0.1 mM EDTA) buffer and stored at − 20° C. Using the primers specific for each fusion, PCR amplification was performed in a 50-μL reaction volume in quadruplicate, consisting of 10 μL of 5X Phusion buffer, 1 μL of 10 mM dNTP, 2.5 mL of each primer at 10 mM, 0.5 μL of HotStart Phusion, and 10 ng of cell line DNA. PCR was performed using a Bio-Rad S1000 Thermal Cycler. The thermal cycle was programmed for 30 s at 98° C for initial denaturation, followed by 34 cycles of 10 s at 98°C for denaturation, 30 s at 59°C for annealing, 30 s at 72°C for extension, and 5 min at 72°C for final extension. Human mixed genomic DNA (Promega, Madison, WI) and no template were used as negative controls. PCR products were purified using Nucleospin Gel and PCR cleanup as per the manufacturer’s instructions (Macherey-Nagel, Duren, Germany). PCR products were then subjected to Sanger sequencing using the Applied Biosystems 3730xl DNA Analyzer as per manufacturer’s instructions (Thermo Fisher, Waltham, MA). Output was compared to original candidate fusion sequence and confirmed.