Illumina HiSeq 2500 paired end sequencing; Loss of Spry1 reduces growth of BRAFV600E-mutant cutaneous melanoma and improves response to targeted therapy
Loss of Spry1 reduces growth of BRAFV600E-mutant cutaneous melanoma and improves response to targeted therapy
Sample 3_p
Cell cultures were established from metastatic lesions surgically removed from cutaneous CM patients that did not underwent prior BRAFi therapy who were referred to the National Cancer Institute of Aviano (Italy), as previously described [1]. Cell cultures were grown in RPMI 1640 Medium, supplemented with 2mM L-glutamine (Sigma-Aldrich) and 10% heat-inactivated fetal calf serum (FCS, Lonza). The identity of paired Spry1KO clones and parental cells was confirmed by short tandem repeat profiling using the Power Plex 1.2 kit (Promega). Stock solutions of UO126 (Sigma), vemurafenib (PLX-4032, SeleckBio), trametinib (GSK1120212, SeleckBio) were prepared in cell-culture grade DMSO (Sigma) as per manufacturer's indications. The study was approved by the Internal Review Board of the Centro di Riferimento Oncologico, IRCCS-National Cancer Institute, Aviano, Italy (IRB number 07-2017). In details , indexed libraries were prepared using 1 ug of total RNA as starting material, with TruSeq Stranded Total RNA  Sample Prep Kit (Illumina Inc.). Libraries were sequenced (paired-end,2x100 cycles) at a concentration of 8 pM/lane on the HiSeq 2500 platform (Illumina Inc). In details , indexed libraries were prepared using 1 ug of total RNA as starting material, with TruSeq Stranded Total RNA  Sample Prep Kit (Illumina Inc.). Libraries were sequenced (paired-end,2x100 cycles) at a concentration of 8 pM/lane on the HiSeq 2500 platform (Illumina Inc).