|
| Status |
Public on Aug 17, 2010 |
| Title |
T00320763 |
| Sample type |
RNA |
| |
|
| Source name |
085
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
age: 56-80 days
tissue region: Hcrt
region name: Hypocretin area
condition: W
specimen: 311-0145
time of day: ZT18
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Laser microdissection using the Leica LMD6000 was carried out on tissue sections (10 um) lightly stained with cresyl violet. Tissue was isolated into RLT buffer (Qiagen) and purified using the RNeasy kit (Qiagen). Using 5 ng total RNA as starting material, a two-round RNA amplification (Ambion MessageAmp II kit) was used to produce sufficient cDNA for biotin-labeling and hybridization to microarrays.
|
| Label |
biotin
|
| Label protocol |
Following the second round of reverse transcription, second-strand cDNA synthesis, and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase in the presence of Biotin-11-UTP. Purified cRNA (10 µg) was fragmented to uniform size (~0.1 kb).
|
| |
|
| Hybridization protocol |
Samples were applied to CodeLink Mouse Whole Genome Bioarrays (formerly supplied by GE Healthcare, Piscataway, NJ; now supplied by Applied Microarrays, Inc, Tempe, AZ) in hybridization buffer. Arrays were hybridized (37°C for 18 h) in a shaking incubator, washed in 0.75X Tris-buffered saline (pH 7.6) at 46°C for 1 h, and stained with Cy5-Streptavidin dye conjugate for 30 min.
|
| Scan protocol |
Rinsed and dried arrays were scanned with a GenePixTM 4000B scanner [Molecular Devices, (formerly Axon Instruments), Sunnyvale, CA] at 5 µm resolution.
|
| Description |
sample barcode: T00320763
|
| Data processing |
Codelink Expression Analysis software (Applied Microarrays, Inc, Tempe, AZ) was used for gridding and feature intensity extraction from the scanned array images. quantile normalization followed by COMBAT
|
| |
|
| Submission date |
Aug 13, 2010 |
| Last update date |
Aug 16, 2010 |
| Contact name |
Changkyu Lee |
| E-mail(s) |
changkyul@alleninstitute.org
|
| Phone |
(206)548-7085
|
| Organization name |
Allen Institute for Brain Science
|
| Street address |
551 N 34th St
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98103 |
| Country |
USA |
| |
|
| Platform ID |
GPL2897 |
| Series (1) |
| GSE23628 |
Sleep Deprivation and Recovery Sleep for 7 Regions of Mouse Brain. |
|
