U87MG cells were maintained in Minimum Essential Medium (MEM; Sigma, St. Louis, MO) supplemented with 10% FBS, 1% non-essential amino acid (Gibco; Thermo Fisher Scientific, Waltham, MA), 1 mM sodium pyruvate (Nacalai Tesque, Kyoto, Japan), and 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen; Thermo Fisher Scientific) in humidified air atmosphere containing 5% CO2 at 37 °C. P4E8 cells were cultured as floating sphere in neurosphere medium (DMEM/F12 supplemented with 1% N2 Max Media Supplement (R&D Systems, Minneapolis, MN), 2% B27 Supplement Minus Vitamin A (Thermo Fisher Scientific, Waltham, MA), 3 mM sodium bicarbonate, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml bFGF and EGF) in humidified air atmosphere containing 5% CO2 at 37 °C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from U87MG and P4E8 cells with TRIzol reagent (Ambion, Thermo Fisher Scientific) and purified using the PureLink RNA Mini Kit (Ambion, Thermo Fisher Scientific). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer series II (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy mini spin column purification (QIAGEN, Valencia, CA). cRNA yield and quality were checked with the NanoDrop ND-1000 Spectrophotometer and Agilent 2100 Bioanalyzer series II.
Hybridization protocol
600 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE 8x60K Microarray Ver2.0 (Agilent) for 17 hours at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute at 37°C with GE Wash buffer 2 (Agilent), then dried.
Scan protocol
Slides were scanned by Agilent Technologies Microarray Scanner (Scan Resolution 3 μm, TIFF file dynamic range 20 bit).
Description
Gene expression in P4E8 cells
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent). Extracted data were 75% tile-normalized and baseline transformation was performed to the median of all samples by Genespring GX software (Agilent Technologies). After removal of control spots, entities where at least 100% of the samples in any 1 out of 2 conditions have values within range were used in the analysis.
