Illumina NovaSeq 6000 paired end sequencing; RNA-Sequencing of vemurafenib-sensitive and resistant melanoma cell line SKMEL-239
RNA-Sequencing of vemurafenib-sensitive and resistant melanoma cell line SKMEL-239
Vem_Resistant_1_p
For culturing the human cell-line model (SK-MEL-239) established by Poulikakos et al. 2011 (Poulikakos et al., 2011) the culturing conditions described in the original publication were used. Parental SK-MEL-239 cells were cultured in 75 cm2 Greiner flasks containing RPMI 1640 medium [Gibco] supplemented with FBS (10X), penicillin/streptomycin (1X) and L-glutamine (1x). Resistant SK-MEL-239 cells used the same culturing media supplemented with 2µM vemurafenib (PLX4032) [SelleckChem]. Parental and resistant cell lines were grown at 37°C with 95% air and 5% carbon dioxide (CO2) environment to maintain physiological pH. Depleted medium was replaced with fresh pre- warmed media every two to three days. When confluency of cells reached approximately 90 to 95 percent, the cells were passaged and stored. To harvest cells, they were detached by adding 0.5 ml/10cm2 of Trypsin-EDTA [Gibco] to the flask area, incubated at 37°C for 1 minute before splitting. Then, trypsin was inactivated by resuspending cells with 9.5 ml/cm2 fresh pre- warmed media. The cell suspension was then harvested by centrifugation at 300 relative centrifugal force (RCF) for 5 minutes at room temperature.  Total RNA was extracted using RNeasy Mini Kit [QIAGEN] according to the manufacturer's instructions. DNA was removed using DNA-free™ DNA Removal Kit [Applied Biosystems] according to the manufacturer's protocol. Poly A selection - NEB Next® Ultra™ RNA Library Prep Kit