|
| Status |
Public on Jan 10, 2010 |
| Title |
wt_3hTNF_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
p65 wt MEFs, 3h TNF, replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Mouse embryonic fibroblasts
|
| Treatment protocol |
Cells were starved overnight before either left untreated or stimulated with 30 ng/ml mouse TNFα for 3 hours.
|
| Growth protocol |
Cells were maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin/streptomycin and non-essential amino acids.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from three biological replicates per sample was isolated at different days using the ‘Total RNA isolation mini kit’ (Agilent Technologies) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
RNA was labeled using the ‘Agilent Low RNA Input Linear Amplification Kit PLUS’ (Agilent Technologies) following the manufacturer’s protocol. Cy3-labeled cRNA was purified with the RNeasy kit (Qiagen). Dye incorporation was assessed with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 25x Agilent fragmentation buffer and 10x 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 Mouse Genome Oligo 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 brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the DNA Microarray-Scanner with Surescan High-Resolution Technology (Agilent Technologies) using one color scan setting for 4x44K array slides (Scan Area 61x21.6 mm, Scan resolution 5um, eCtended Dynamic range selected, Dye channel is set to Green, Green PMT XDR Hi is set to 100% and Green PMT XDR Lo to 10%).
|
| Description |
Gene expression after 3 hours of TNFalpha stimulation from p65(-/-) MEFs genetically complemented with p65 wild type
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1_v5_91_0806 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. Data was normalized with default settings using ‘Rosetta Resolver® Gene Expression Data Management and Analysis System’ (Rosetta Biosoftware).
|
| |
|
| Submission date |
Mar 11, 2009 |
| Last update date |
Jan 02, 2010 |
| Contact name |
Karin Rothgiesser |
| Organization name |
Institute of Veterinary Biochemistry and Molecular Biology
|
| Street address |
Winterthurerstrasse 190
|
| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE15196 |
Acetylation of p65 at lysine 314 is important for late NF-k(kappa)B-dependent gene expression |
|
