Comment[ArrayExpressAccession] E-GEOD-43946 MAGE-TAB Version 1.1 Public Release Date 2015-01-21 Investigation Title Human induced pluripotent stem cells reveal early developmental molecular correlates with a probable Leber congenital amaurosis type I Comment[Submitted Name] Human induced pluripotent stem cells reveal early developmental molecular correlates with a probable Leber congenital amaurosis type I Experiment Description This SuperSeries is composed of the SubSeries listed below. Refer to individual Series Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name de la Grange Person First Name Pierre Person Email pierre.delagrange@genosplice.com Person Affiliation GenoSplice technology Person Address GenoSplice technology, iPEPS - ICM - Hopital de la Pitie Salpetriere - 47-83 bd de l'Hopital, Paris, France Person Roles submitter Protocol Name P-GSE43946-1 P-GSE43946-4 P-GSE43946-5 P-GSE43946-7 P-GSE43946-2 P-GSE43946-3 P-GSE43946-6 Protocol Description 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 ID_REF = VALUE = Quantile gene and normalized gene level expression values (log2) from EASANA (GenoSplice technology) Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix) 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 200ÎĽl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001 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 Retinal Pigmented Epithelial (RPE) cells from iPSCs using the procedure schematically summarized. When hiPSCs on feeders reached confluence, medium was removed and cells were cultured with DMEM + GlutamaxII (High Glucose) containing 20% KnockOutTM SR (both from Invitrogen) without FGF2. Pigmented areas begun to appear within 2 weeks and allowed to expand until they reached a few millimetres (one more week). They were mechanically cut under a stereomicroscope using a needle and then placed on 1:30 Matrigel (BD Matrigel™ Basement Membrane Matrix, BD Biosciences, France) coated plate. When the confluence reached 100%, cells were dissociated in 0.05% Trypsin-EDTA and seeded on Matrigel at a density of 100,000 cells/cm2. They were then cultured in DMEM + GlutamaxII (High Glucose) containing 4% KnockOutTM SR (both from Invitrogen). 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. Total RNA was extracted with the RNeasy Mini kit, according to manufacturer’s instructions. Affymetrix Gene ChIP Scanner 3000 7G Protocol Type normalization data transformation protocol labelling protocol hybridization protocol growth protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name cell line cell type passage genotype sex Experimental Factor Type cell line cell type passage genotype sex Publication Title Human induced pluripotent stem cells as a tool to model a form of Leber congenital amaurosis. Publication Author List Lustremant C, Habeler W, Plancheron A, Goureau O, Grenot L, de la Grange P, Audo I, Nandrot EF, Monville C PubMed ID 23663011 Publication DOI 10.1089/cell.2012.0076 Comment[SecondaryAccession] GSE43946 Comment[GEOReleaseDate] 2015-01-21 Comment[ArrayExpressSubmissionDate] 2013-01-31 Comment[GEOLastUpdateDate] 2015-01-22 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-43946.sdrf.txt