|Public on May 22, 2012
|hESC NSC 2
|neurosphere from hESC H9
|sample type: neurosphere from hESC H9
cell line: H9
treatment: hESC media lacking bFGF with TGF-b inhibitor and Noggin
|hESC cultures were disaggregated using accutase for 10 min and plated on Matrigel-coated dishes in mTESR medium. hESC were allowed to expand for 3 d or until they were nearly confluent. The differentiation towards neural progenitors was performed in hESC media lacking bFGF with 10 µM TGF-b inhibitor (Tocris) and 500 ng/ml of Noggin (R&D). Upon day 5 of differentiation, the TGF-ß inhibitor was withdrawn while maintaining 500 ng/ml of Noggin and increasing amounts of N2 media (25%, 50%, 75%) was added for further 6 days with changing media for every 2 d.
|RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit.
|A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip Human Gene 1.1 ST Array Plate followed by staining and washing in the GeneTitan® Instrument (Affymetrix) according to the manufacturer's procedures.
|To assess the raw probe signal intensities, chips were scanned using the GeneTitan® HT Array Plate Scanner (Affymetrix).
|neurospeheres derived from hESC
|RMA-normalized expression values as obtained with the xps package (version 1.10.2) of BioConductor
|Jun 07, 2011
|Last update date
|May 23, 2012
|Herestraat 49 Box 816
|Transduction of human fibroblasts with Zic3 combined with OCT4, SOX2 and KLF4 induces stable neural progenitor cell lines