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Sample GSM379309 Query DataSets for GSM379309
Status Public on Jan 10, 2010
Title K314/315R_3hTNF_rep2
Sample type RNA
 
Source name p65 K314/315R 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 K314/315R mutant
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

Data table header descriptions
ID_REF
VALUE Normalized signal intensity

Data table
ID_REF VALUE
A_52_P136751 300.3877
A_52_P1156465 608.38086
A_52_P661982 11028.364
A_52_P779137 10.02739
A_51_P428086 442.74478
A_52_P271894 41.910748
A_51_P403693 10116.272
(+)E1A_r60_a20 1506.6495
A_52_P812372 9.989136
A_51_P148587 9.761147
A_52_P272936 12.528261
A_51_P405496 44.99178
A_51_P345110 8314.578
A_51_P244234 10.448775
A_52_P205710 19.071447
A_51_P410892 6644.1484
A_51_P258698 6218.601
A_52_P107923 13.795128
A_52_P395685 19.567831
A_51_P127738 48.565777

Total number of rows: 41266

Table truncated, full table size 910 Kbytes.




Supplementary file Size Download File type/resource
GSM379309.txt.gz 6.6 Mb (ftp)(http) TXT
Processed data included within Sample table

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