AB 5500xl Genetic Analyzer paired end sequencing; RNAseq of human Ewing, MSC, fibroblast cell lines - A side population of Ewing cells exhibits stem cell properties
RNAseq of human Ewing, MSC, fibroblast cell lines - A side population of Ewing cells exhibits stem cell properties
Ewing Sarcoma cell lines CADO-ES1, was purchased form DSMZ. All other Ewing sarccoma cell lines were kindly supplied by Franz von Valen. Ewing sarcoma cell lines were cultivated in RPMI1640 medium, supplemented with 10% (v/v) FCS, 1% Glutamin and 1% of an antibiotic cocktail containing penicillin and streptomycin. All Ewing sarcoma cell lines were maintained on cell culture plastic coated with collagen (0,5mg/ml). Mesenchymal stem cells (MSC) were isolated from BM biopsies. Human skin fibroblast (hfib) were obtained from genital skin biopsies. MSCs and hFBs were cultivated on untreated cell culture plastic in DMEM, 10% (v/v) FCS, 1% Glutamin and 1% of an antibiotic cocktail containing penicillin and streptomycin. All cell lines were incubated at 37°C and 5%CO2 in a humidified atmosphere. Routine microbiological analysis was done using a PCR Mycoplasma Detection Kit according to suggested protocols (Applied Biosystems, Darmstadt, Germany). The extraction of nucleic acids from cell cultures was performed by the use of the RNAeasy Kit (Qiagen) according to the supplier's recommendations. For RNA library preparation 500 – 5000ng RNA were used. ERCCs as an additional internal quality control were added to the libraries. We followed exactly the Thermo-Fischer/Life-Technology(ABI) protocols for the SOLID-5500XL Technology for RNAseq. Most of the procedures were run in a core lab facility of Life-Technology as a commercial service.
Experimental Factor: disease
normal
Experimental Factor: genotype
wild type genotype
Experimental Factor: cell type
fibroblast of dermis
Experimental Factor: cell line
not applicable