Illumina HiSeq 4000 sequencing; Whole-exome sequencing of genomic DNA from patient-derived melanoma cell lines resistant to either vemurafenib (PLX) or trametinib (TRA).
Whole-exome sequencing of genomic DNA from patient-derived melanoma cell lines resistant to either vemurafenib (PLX) or trametinib (TRA).
Melanoma specimens were obtained during surgical intervetions, and grown in vitro in serum-free EGF/bFGF-containing medium as described previously (Sztiller-Sikorska et al., 2012 Melanoma Research). Cell resistance was developed for 10uM vemurafenib or 50 nM trametinib. DNA was extracted using DNeasy Blood & Tissue Kit from Qiagen according to manufacturer protocol.  DNA samples were quantified using Picogreen (Invitrogen Thermo Fisher Scientific, Carlsbad, CA, USA) and resolved by 1% agarose gel electrophoresis (30 min, 100 V) to confirm the presence of high molecular weight fragments. DNA samples were then prepared according to an Agilent SureSelect Human All Exome V6 kit (Agilent Technologies) which is a solution-based system utilizing ultra-long 120-mer biotinylated cRNA baits to capture regions of interest. Targeted regions were selected using magnetic streptavidin beads, amplified and loaded on the sequencer.
Experimental Factor: compound
trametinib
Experimental Factor: dose
50