Mitochondrial superoxide dismutase controls metabolic plasticity in pancreatic cancer
Mitochondrial superoxide dismutase controls metabolic plasticity in pancreatic cancer
Cells were seeded at a density of 10.000 cells in 6 cm dishes and after attachment, were cultured for 24 hours. Culture in standard medium +/- 60 µM 10058-F4 (Myc Inhibitor) Total RNA of cancer cell lines was isolated using Maxwell® 16 LEV simplyRNA Tissue Kit (AS1280, Promega) and Maxwell® 16 Instrument (Promega) as per manufacturer's instructions Library preparation for bulk 3'-sequencing of poly(A)-RNA was done as described previously (Parekh et al., 2016) barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 5' ends of the cDNAs were extended by a template switch oligo (TSO) and after pooling of all samples full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3'-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs.