Genomic DNA Extraction and Sequencing

Genomic DNA was isolated either from the indicated cell lines or from formalin-fixed paraffin-embedded nevi, primary or metastatic melanoma patient samples. For melanoma cell lines, cell pellets were incubated at 50°C overnight in 100 mM NaCl, 10 mM Tris–HCl, pH8, 25 mM EDTA, 0.5 SDS and 0.1 mg/ml Proteinase K and the genomic DNA was extracted with phenol-chloroform. For paraffin-fixed tissues, tissue sections scraped from five 10-μm samples were incubated at 60°C overnight (in SSC buffer, 180 mM NaCl, 0.45% SDS, 2 mg/ml Proteinase K and 1 mM DTT) and the DNA was extracted with phenol–chloroform. A 227 bp fragment including pre-miR146a was amplified by PCR using the primers listed in Supplementary file 1E 5, cloned using pGEM-T kit (Promega), and plasmid DNA isolated from 24 bacterial colonies were sequenced. Similarly, fragments of BRAF and NRAS were amplified for genotyping using primers listed in Supplementary file 1E from genomic DNA isolated from patient-derived samples, cloned using pGEM-T kit (Promega), and plasmid DNA isolated from 24 bacterial colonies were sequenced using S6 primers. All the sequencing was performed using Sanger sequencing method that typically have the error rate that range from 0.001 to 1% (Hoff, 2009 (link)).