Bigdye v1

BRAF, NRAS, and DUSP4 cDNA levels were quantified by real time RT-PCR using TUBULIN and GAPDH levels for normalization. Relative expressions were calculated using the delta-Ct method. BRAF, NRAS, and DUSP4 gDNA relative copy numbers were quantified by real time PCR with total gDNA content estimated by assaying the β-globin gene in each sample. All primer sequences are available upon request. Sanger sequencing was performed using purified PCR via BigDye v1.1 (Applied Biosystems) in combination with a 3730 DNA Analyzer (Applied Biosystems). WES of M249 triple cell lines were analyzed for shared and distinct genetic alterations and their phylogenetic relationship.

DNA was extracted from frozen tumor tissue and blood with Maxwell 16 Tissue and Blood DNA purification kits (Promega, Madison, WI, USA). Microdissection of tumor and normal tissue was performed manually on formalin fixed and paraffin embedded tissue slides and DNA was extracted with Maxwell 16 FFPE Tissue LEV DNA purification kit (Promega) after 16 h incubation with proteinase K at 65°C.
The entire mitochondrial genome was sequenced with the MitoAll kit (Applied Biosystems, Foster City, CA, USA) (10 (link)). PCR reactions were performed in 10 µl with KAPA2G Fast Readymix on 10 ng of DNA. PCR amplicons were purified by adding 1U of FastAP Thermosensitive Alkaline Phosphatase and 0.5 U of Exonuclease I to each PCR reaction and incubation at 37°C for 30 min, followed by heat inactivation at 80°C for 15 min. Sequencing was performed with BigDye v1.1 (Applied Biosystems) according to the manufacturer’s instructions. The reactions were cleaned up with Performa DTR Plates (Edge Bio) and run in an ABI 3730 Genetic Analyzer automated sequencing machine (Applied Biosystems). Electropherograms were inspected and aligned to the human mitochondrial reference sequence (NC_012920) using Sequencher 4.10.4 (Genecodes, Ann Arbor, MI, USA). All sequences were submitted to the NCBI database (KJ801401 – KJ801483).