|
| Status |
Public on Apr 13, 2007 |
| Title |
Day 5_M_-LIF_Bchip |
| Sample type |
RNA |
| |
|
| Source name |
embryonic stem cell, medium Oct4 subpopulation, 5 days following LIF withdrawal
|
| Organism |
Mus musculus |
| Characteristics |
R1 cell line
|
| Treatment protocol |
Oct4:eGFP ESCs were plated on 10-cm tissue culture treated dishes (Falcon) coated with 0.1% gelatin in differentiation media at a density of 0.5x106 cells/dish for 5 days of differentiation, 106 cells/dish for 3 days of differentiation, 2x106 cells/dish for 2 days of differentiation or 3.5x106 cells/dish for 1 day of differentiation. Control, cells were harvested two days after plating in +LIF media. Differentiating cells were FACS sorted by eGFP expression following trypsinization and resuspension in 2% FBS in PBS at a dilution of 8x106 cells/mL.
|
| Growth protocol |
R1 ESCs and Oct4:eGFP ESCs (Viswanathan et al., 2003) were cultured at 37°C and 5% CO2, on a layer of mitomycin-treated embryonic fibroblasts (MEFs) in ESC media consisting of Dulbecco modified eagle serum (DMEM) supplemented with 15% FBS (North Bio, Lot SF30408), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine (all from Gibco), 1000 U/mL leukemia inhibitory factor (LIF) (ESGRO, from Chemicon, batch 11061065) and 100 µM beta-mercaptoethanol (Sigma). Differentiation media consisted of either ESC media without LIF, or ESC media without LIF and supplemented with 0.1 uM retinoic acid. Selection media consisted of ESC media supplemented with 150 g/mL G418 (Gibco). ESCs were passaged every two days at a ratio of 1:5 by washing with PBS (Gibco), dissociating with 0.05% trypsin (Gibco) for 5 minutes at 37°C and resuspending in ESC media. Media was changed daily.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
The protocol was modified from the Affymetrix GeneChip technical note entitled: Eukaryotic Small Sample Target Labeling. 500 ng of cRNA was used for the second cycle of amplification and 1 g of purified cDNA from the second cycle was used for biotin labeling. It was determined that this resulted in optimized linearity of amplification. Amplified and biotin labeled cRNA was hybridized to both the Affymetrix GeneChips MG_U74av2 and MG_U74bv2.
|
| |
|
| Hybridization protocol |
Amplified and biotin labeled cRNA was hybridized to both the Affymetrix GeneChips MG_U74av2 and MG_U74bv2. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
gene expression of 5 day LIF differentiated ESCs
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method.
|
| |
|
| Submission date |
Apr 12, 2007 |
| Last update date |
Apr 12, 2007 |
| Contact name |
William L Stanford |
| E-mail(s) |
[email protected]
|
| URL |
http://www.wlstanfordlab.com/
|
| Organization name |
University of Toronto
|
| Department |
IBBME
|
| Lab |
William L. Stanford
|
| Street address |
164 College St
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3G9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL82 |
| Series (1) |
| GSE7506 |
Prediction and Testing of Novel Networks Regulating Embryonic Stem Cell Self-Renewal and Commitment |
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