|
| Status |
Public on May 10, 2009 |
| Title |
H1hESC 3h 20%O2 |
| Sample type |
RNA |
| |
|
| Source name |
human embryonic stem cells, BMP4 3h, 20% O2
|
| Organism |
Homo sapiens |
| Characteristics |
human embronic stem cell, NIH code:WA01
|
| Treatment protocol |
hESC were cultured in either a standard humidified tissue culture CO2 incubator, set at 5% CO2 in air, or a sealed chamber (MIC-101, Billups-Rothenberg, Del Mar, CA) filled with the humidified physiological O2 mixture (~4 % O2/5 % CO2/92-90 % N2) in a standard tissue culture incubator at 37°C. Oxygen levels, measured by using a Bacharach CO2/O2 Analyzer (Bacharach, Model 2830), were ~19% in the standard incubator and 3.5–4.5% in the physiological O2 chamber. To minimize temporal ‘oxygen surge’ at each media change and at passage of the cells cultured under physiological O2 condition, the medium was pre-equilibrated for 24 h under physiological gas mixture. Processing cells while outside the incubator is generally completed within 30 min to minimize periodic reperfusion effects. The H1 hESC lines had been maintained in a physiological O2 atmosphere since passage 26. Total RNA was isolated after 0, 3, 12, 24, 72, and 120 h of BMP4 exposure at passage number 37 and had been maintained on Matrigel™ for 2 passages.
|
| Growth protocol |
Human ESC H1 (WA01) at passage 23 was purchased from WiCell Research Institute (Madison, WI) and cultured in six-well tissue culture plates on a monolayer of γ-irradiated (8,000 cGy) mouse embryonic fibroblast (MEF) feeder cells until passage number 35. Thereafter, cells were cultured on a substratum of 1:30 dilutions of Matrigel™ in medium conditioned by MEF. The cells were used in the experiemts at passage number 37.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from hESCs by using RNA STAT-60 reagent (Tel-Test Inc, Friendswood, TX) according to the manufacturer's instructions. Isopropanol precipitated RNA pellets were dissolved in 35 ul of nuclease-free water and purified with spin columns of RNeasy Mini Kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Biotin labeling
|
| |
|
| Hybridization protocol |
cRNA was fragmented to uniform size and verified on the Bioanalyzer. Agilent Whole Genome arrays were hybridized with the cRNA target
|
| Scan protocol |
Slides were washed and scanned on an Agilent G2565 Microarray Scanner.
|
| Description |
The cRNA target was hybridized to Agilent-014850 Whole Human Genome Microarrays 4x44K (G4112F). Slides were washed and scanned on an Agilent G2565 Microarray scanner, and data analyzed with Agilent Feature Extraction and GeneSpring GX v7.3.1 softwares.
|
| Data processing |
Data was analyzed with Agilent Feature Extraction and GeneSpring GX v7.3.1 softwares.
|
| |
|
| Submission date |
Feb 11, 2008 |
| Last update date |
Feb 09, 2009 |
| Contact name |
Toshihiko Ezashi |
| E-mail(s) |
ezashit@missouri.edu
|
| Phone |
573 884-9601
|
| Organization name |
University of Missouri-Columbia
|
| Department |
Animal Sciences
|
| Lab |
R.M. Roberts
|
| Street address |
240a CS Bond Life Sciences Center
|
| City |
Columbia |
| State/province |
MO |
| ZIP/Postal code |
65211 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE10469 |
Time course progression of BMP4-treated hESC gene expressions under 4 % and 20 % oxygen conditions. |
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