Graded brain tumor specimens were obtained with informed consent, as part of a study protocol approved by the SingHealth Centralised Institutional Review Board A. GPC lines were described in our previous work. Briefly, cells were seeded as free-floating spheres at a density of 2,500 cells per cm2 in chemically defined serum-free growth medium containing basic fibroblast growth factor (bFGF; 20 ng/ml; PeproTech, New Jersey), epidermal growth factor (EGF; 20 ng/ml; PeproTech), heparin (5 μg/ml; Sigma-Aldrich, St Louis), and serumfree supplement (B27; 1x; Gibco, Grand Island, NY) in a 3:1 mix of Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) and Ham’s F-12 Nutrient Mixture (F-12; Gibco). Neurospheres were mechanically triturated with a flame-drawn glass Pasteur pipette twice weekly and re-seeded in fresh culture medium
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from tumor neurospheres using TRI Reagent (Molecular Research Center, Cincinnati, OH). Thereafter, RNA samples were hybridized to Illumina HumanRef-8 v2 Expression Beadchip Array.
Label
biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina HumanRef-8 v2 Expression Beadchip
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Data processing
The data were normalised using quantile normalisation on exonic probesets with lumi packages in R
