Human iPS cells were expanded on-to mitomycin-C inactivated Zenith Mouse Embryonic Fibroblast (MEF) layer feeders (IVFonline.com). Cells were cultured using DMEM/F12 (1:1) Glutamax medium supplemented with 20% KnockOutTM SR, 1X MEM non-essential amino acids, 0.05 mM 2-mercaptoethanol (all from Invitrogen) and 10 ng.ml-1 FGF2 (PeproTech, Rocky Hill, NJ, USA). We differentiated neural stem cells from iPSCs using a multistage differentiation protocol. The procedure is schematically summarized. To induce Neuro EPithelial cells (NEP) formation, clumps were seeded on 0.0015% poly-L-ornithine (PO, Sigma Aldrich)/2 µg.ml-1 laminin (Invitrogen) coated culture dishes and cultured using DMEM/F12 (1:1) Glutamax medium containing Neurobasal medium (1:1) supplemented with 2% B27 supplement without vitamin A, 1% N2 supplement, 0.05 mM 2-mercaptoethanol (all from Invitrogen), 5 ng.ml-1 FGF2, 300 ng.ml 1 human Noggin (both from PeproTech) and 20 µM SB431542 (Tocris Bioscience, Bristol, United Kingdom) until appearance of NEP (8 days of differentiation). To obtain NSC, NEP were cultured using DMEM/F12 (1:1) Glutamax medium containing Neurobasal medium (1:1) supplemented with 2% B27 supplement without vitamin A, 1% N2 supplement, 0.05 mM 2-mercaptoethanol (all from Invitrogen) supplemented by 10 ng.ml-1 human EGF (Epidermal Growth Factor), 20 ng.ml-1 human BDNF (Brain-Derived Neurotrophic Factor) (both from R&D Systems, Minneapolis, MN, USA) and 10 ng.ml-1 FGF2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the RNeasy Mini kit, according to manufacturer’s instructions.
Label
biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
Hybridization protocol
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 200μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001
Scan protocol
Affymetrix Gene ChIP Scanner 3000 7G
Description
NSC_p07_4603cl27_p47
Data processing
Data were processed using EASANA from GenoSplice technology. GC background correction were applied to probe intensities (core), gene level expression were summarized from the corrected intensities and then quantile normalized. probe group file: HuEx-1_0-st-v2.r2.pgf meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps