|
| Status |
Public on Mar 05, 2009 |
| Title |
LIF_rep1_Cy3 |
| Sample type |
RNA |
| |
|
| Source name |
R1 mESC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryo stem cells
cell line: R1
|
| Treatment protocol |
Independent quadruplicate: groupA in LIF (Chemicon) without feeders, groupB in 0.2mM sodium butyrate (without LIF) for 15 passages
|
| Growth protocol |
mESC in DMEM, 15% serum replacer, glutamax, pen/strep, NEAA, pyruvate (Invitrogen) and mercaptoethanol (Sigma), grown on gelatin (Sigma) at 37C in 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction utilized the Rneasy Minikit (Qiagen) following manufacturer's protocol. The RNA was treated with Dnase and given to the Center for Array Technologies for quantitation and purity analysis.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) and Cyanine-5 (Cy5) labeled cRNA was prepared from 0.5 ug RNA using the Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
825ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) and 825ng of Cy5-labelled cRNA (specific activity >10.0 pmol Cy5/ug cRNA) were combined and fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) and Agilent Whole Genome Mouse Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by compressed nitrogen (N2) gun.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Red and both Green PMT and Red PMT arer set to 100%).
|
| Description |
Gene Expression in LIF
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE2-v5_95 and Grid: 014868_D_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Mar 04, 2009 |
| Last update date |
Mar 04, 2009 |
| Contact name |
Brig Mecham |
| E-mail(s) |
bmecham@fhcrc.org
|
| Organization name |
University of Washington
|
| Street address |
1100 Fairview Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
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