cultured for two passages on matrigel in mTESR medium
Growth protocol
mTESR medium
Extracted molecule
total RNA
Extraction protocol
Rneasy (Qiagen)
Label
biotin
Label protocol
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.
Hybridization protocol
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.
Scan protocol
To assess the raw probe signal intensities, chips were scanned using the GeneTitan® HT Array Plate Scanner (Affymetrix).
Description
hESC
Data processing
RMA-normalized expression values as obtained with the xps package (version 1.10.2) of BioConductor
