The iPSCs were cultured under the feeder-free and serum-free condition with AK03N medium (Ajinomoto) on the 6-well culture plate coated with iMatrix-511 (Nippi). After the undifferentiated iPSCs reached confluence in culture, the iPSCs were dissociated with Accutase (Millipore) and seeded first on the 3-D culture plate (EZSPHERE®, AGC TECHNO GLASS) at a density of 2×10^5 cells/mL in embryoid body (EB) induction medium which composed from DMEM/F12 (Sigma-Aldrich) supplemented with 1% N-2 (GIBCO), 2% MACS® NeuroBrew-21 (Miltenyi Biotec), 2 mM L-glutamine (Sigma-Aldrich), 20 ng/ml recombinant human epidermal growth factor (EGF: PeproTech), 1% penicillin/streptomycin (GIBCO) and 10 μM Y-27632 (Wako). The spent medium was changed once on EB culture day 4 and uniformly sized EBs were formed in 7 days. Subsequently, the EBs were replated on non-coated 10 cm dish in NCCs induction medium, DMEM/F12 with 1% N-2, 2 mM L-glutamine, 1% penicillin/streptomycin, 20 ng/ml EGF, 20 ng/ml recombinant human fibroblast growth factor-basic (bFGF: Wako), and 5 μg/ml heparin (Sigma-Aldrich). To allow the EBs to attach the culture dish and induced NCCs to migrate from the attached EBs, the culture dish was stand for first 3 days and the spent medium was changed on days 4, 8, 11, 13, and 14 after re-plating of the EBs. NCCs induction was completed on day 14. .
Extracted molecule
total RNA
Extraction protocol
The total RNA of iPSCs (n=2) and iPSC-NCCs (n=2) were extracted by High Pure RNA Isolation Kit (Roche) after the cells were harvested.
Label
biotin
Label protocol
We followed protocol of manual that is GeneChip® WT PLUS Reagent Kit User Manual.
Hybridization protocol
We followed protocol of manual that is GeneChip® WT PLUS Reagent Kit User Manual.
Scan protocol
We followed manufacturer's protocol, and used GeneChip® Scanner 3000 7G.
Description
iPS 2
Data processing
Data acquisition and normalization (SST-RMA) were used Affymetrix® Expression Console™ Software.
