Protocols: 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
ENA first public
2022-04-16
ENA last update
2022-04-16
External Id
SAMEA13959307
INSDC center alias
Systems Biology Ireland University College Dublin
INSDC center name
Systems Biology Ireland University College Dublin
INSDC first public
2022-04-16T00:27:40Z
INSDC last update
2022-04-16T00:27:40Z
INSDC status
public
Submitter Id
E-MTAB-11609:Parental_4
broker name
ArrayExpress
cell line
SK-MEL-239
cell type
melanocyte
common name
human
developmental stage
adult
disease
melanoma
genotype
BRAF V600E
growth condition
media
organism part
skin of body
sample name
E-MTAB-11609:Parental_4
strain
parental