|
| Status |
Public on Jul 27, 2010 |
| Title |
P0.5_KO_Fr_4 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse E14.5 neocortices and Postnatal day (P) 0.5 brains
|
| Organism |
Mus musculus |
| Characteristics |
strain: CD1
developmental stage: P0.5
genotype/variation: Tbr1 -/-
tissue: frontal brain
|
| Biomaterial provider |
The University of Washington (Seattle, WA).
|
| Growth protocol |
Tbr1 mutant mice were maintained as heterozygotes on CD1 background. To generate Tbr1 homozygous null animals, heterozygous males were crossed with heterozygous females. The day of the vaginal plug was considered embryonic day (E) 0.5. Pregnant dams were deeply anesthetized with Avertin (3-3-tribromoethanol; Sigma) and killed by cervical dislocation, after which embryos were harvested. For immunological analysis or in situ hybridization, embryos were rapidly removed and perfused with 4% paraformaldehyde in 1×PBS (pH 7.4). Embryonic brains were postfixed by immersion in cold buffered (4°C) 4% paraformaldehyde for 4-16 h. Brains were cryoprotected in 30% sucrose in 0.1 M sodium phosphate (pH 7.0), frozen in optimum cutting temperature compound (Sakura Finetek), cryosectioned at 12 um, andmounted on Superfrost Plus slides (Thermo Fisher Scientific). Slides were stored at -80°C until needed. All experimental procedures were approved by the University of Washington and Seattle Children's Research Institute Institutional Animal Care and Use Committees.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA purification was performed as described (PMID: 19380167). Briefly, tissues were dissolved by low-power sonication in a solution consisting of 4 M guanidinium thiocyanate and 0.1 M 2-mercaptoethanol. After sonication, sodium acetate (pH 4), phenol (pH 4), and a 24:1 mix of chloroform and isoamylic acid were added on ice; after vigorous vortexing, aqueous solution containing total RNA was separated by high-speed centrifugation at 4 °C for 20 min. RNA was precipitated by adding an equal volume of isopropanol, and it was stored at -20 °C overnight and then, pelleted by centrifugation for 20 min at maximum speed at 4 °C, rinsed with 70% ethanol, and resuspended in an appropriate volume of nuclease-free water.
|
| Label |
biotin
|
| Label protocol |
For microarray hybridization, RNA was processed for hybridization to Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays through a core facility in the Functional Genomics Laboratory at the Center for Ecogenetics and Environmental Health at the University of Washington according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
The standard hybridization protocol was used as recommended by Affymterix.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 through the University of Washington Functional Genomics Laboratory at the Center for Ecogenetics and Environmental Health (http://depts.washington.edu/ceeh/ServiceCores/FC1/FC1.html).
|
| Description |
Mouse E14.5 neocortices and Postnatal day (P) 0.5 brains.
|
| Data processing |
Affymetrix Expression Console v1.1 RMA normalization log2
|
| |
|
| Submission date |
Jun 15, 2010 |
| Last update date |
Sep 05, 2017 |
| Contact name |
James William MacDonald |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
Environmental and Occupational Health Sciences
|
| Street address |
4225 Roosevelt Way NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105-6099 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE22371 |
Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex |
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