|
| Status |
Public on Apr 15, 2008 |
| Title |
Sample 306b, cell line 306 |
| Sample type |
RNA |
| |
|
| Source name |
embryonic stem cell line 306
|
| Organism |
Macaca mulatta |
| Characteristics |
parent ID M106F244
|
| Treatment protocol |
Non-human primate ESCs were isolated by scraping colonies with good morphology
|
| Growth protocol |
Inner cell mass (ICM) cells were isolated from expanded blastocysts using immunosurgery. First, the zona pellucidas were removed by exposure to acid Tyrode’s media (Specialty Media, Phillipsburg, NJ) for 30-45 seconds. The ICM was next isolated by exposure first to anti-monkey antiserum [1:3, Sigma, St. Louis MO] for 15-20 min at 37° C and then transferred to guinea pig complement [1:3; Biomeda, Foster City, CA] for an additional 15-20 min at 37°C. This destroys the outer trophectoderm, leaving the ICM cells intact. Mechanical pipetting with a 75 ìm tip was used to dissociate the ICM, prior to washing and plating onto mitomycin C-treated mouse embryonic fibroblasts (MEF; Specialty Media) in 80% Knockout Media; 20% Knockout Serum Replacer; 1mM L-glutamine; 0.1mM Nonessential Amino Acids; 1% Penicillin/Streptomycin; 12ng/mL of bFGF; 10ng/mL of Activin A and 10ng/mL hLIF (All components from Invitrogen except hLIF, Chemicon and Activin A, Sigma). After 10 to 14 days, sections of expanded ICM with characterisitic ESC morphology (tightly packed cells with high nuclear:cytoplasm ratio and prominent nucleoli) are dissociated with a fine glass capillary and colony pieces transferred to new MEF for expansion with culture media changed every 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 300 ng total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on Rhesus Macaque Genome 49 format Arrays
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip 3000 Scanner
|
| Description |
Male
|
| Data processing |
The data were analyzed with Affymetrix GCOS software, Statistical analysis was performed using Scoregene gene expression package at the Computational Molecular Biology website of The Hebrew University,and data visualization was performed using Genexpress (Stanford University), Spotfire Decision Site 8.0 (Spotfire Inc. Göteborg, Sweden) and Treeview (Eisen lab).
|
| |
|
| Submission date |
Apr 17, 2007 |
| Last update date |
Apr 17, 2007 |
| Contact name |
Naftali Kaminski |
| Organization name |
University of Pittsburgh Medical Center
|
| Department |
Pulmonary, Allergy and Critical Care Medicine
|
| Lab |
NW 628 MUH
|
| Street address |
3459 5th Avenue
|
| City |
pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
| |
|
| Platform ID |
GPL3535 |
| Series (1) |
| GSE7534 |
Pedigreed Primate Embryonic Stem Cells Express Homogeneous Familial Gene Profiles |
|
