Cells were exposed to TPA at 50nM and were harvested at 0, 1, 3, 6, 12 18, 24, 30, 36, 42, 48, 72, 96, 168 hours after TPA treatment for RNA purification.
Growth protocol
Human ES cells (H9 cell line) were cultured in human ES cell culture medium preconditioned with irradiated mouse embryonic fibroblasts (MEF) on Matrigel (BD Biosciences, Bedford, MA) as described (Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001;19:971-974). For routine maintenance, cells were passaged with 1mg/ml Collagenase (Invitrogen Corporation, Carlsbad, CA). For the TPA treatment time course experiment, cells were individualized with 0.05% Trypsin-EDTA, and seeded at 2 X 106 cells per 10 cm tissue culture dish. Twenty-four hours later, the cells formed small colonies with relatively uniform size (20-30 cells per colony).
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared with RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) and subjected to on-column DNase treatment.
Cells were exposed to TPA at 50nM and were harvested at 0, 1, 3, 6, 12 18, 24, 30, 36, 42, 48, 72, 96, 168 hours after TPA treatment for RNA purification.
Growth protocol
Human ES cells (H9 cell line) were cultured in human ES cell culture medium preconditioned with irradiated mouse embryonic fibroblasts (MEF) on Matrigel (BD Biosciences, Bedford, MA) as described (Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001;19:971-974). For routine maintenance, cells were passaged with 1mg/ml Collagenase (Invitrogen Corporation, Carlsbad, CA). For the TPA treatment time course experiment, cells were individualized with 0.05% Trypsin-EDTA, and seeded at 2 X 106 cells per 10 cm tissue culture dish. Twenty-four hours later, the cells formed small colonies with relatively uniform size (20-30 cells per colony).
Extracted molecule
genomic DNA
Extraction protocol
Total RNA was prepared with RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) and subjected to on-column DNase treatment.
Label
Cy3
Label protocol
Amino Allyl labeling method
Hybridization protocol
Array hybridization, wash were performed according to NimbleGen’s recommended protocol
Scan protocol
Arrays were scanned using a GenePix 4000B scanner and the PMT settings were followed by NimbleGen array manual
Description
the second channel (genomic DNA) serve as common reference across the experiments
Data processing
1. Image extraction: NimbleScan provided by NimbleGen is used to extract signal data from image files (*.TFF). Signal meansurements were saved in .pair files. 2. Normalization: Signal intensities from RNA and gDNA samples are normalized with Robust Multiple-chip Analysis (RMA) algorithm (Irizarry et al., 2003) separately. A median-adjusted ratio is calculated for each gene by dividing its signal intensity from the RNA sample by the sum of the corresponding signal intensity from the gDNA sample and median signal intensity of all genes from the gDNA channel.
Protein Kinase C Mediated Extraembryonic Endoderm Differentiation of Human Embryonic Stem Cells
Data table header descriptions
ID_REF
VALUE
A median-adjusted ratio is calculated for each gene by dividing its signal intensity from the RNA sample by the sum of the corresponding signal intensity from the gDNA sample and median signal intensity of all genes from the gDNA channel.
