|
| Status |
Public on Jan 01, 2013 |
| Title |
NOD2-L1007fsinsC_2h_MDP_biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
HEK-293
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK-293
genotype: NOD2 L1007fsinsC
stimulation: Muramyl-dipeptide (MDP) 10 µg/ml
time point: 2 h
|
| Treatment protocol |
FLP-IN HEK 293-NOD2wt, HEK 293-NOD2L1007fsinsC and HEK 293mock cells were cultivated in medium without FCS for 7 h before stimulation with 10 µg/ml MDP for 0h, 2h and 6h. Cells were harvested simultaneously.
|
| Growth protocol |
NOD2wt and NOD2-L1007fsinsC expressing cells were generated with the FLP-IN system (Invitrogen, Carlsbad, CA, USA). HEK 293 cells containing the recombination site were purchased from Invitrogen. Wildtype and mutated NOD2 were inserted to the genomic recombination site by FLP-recombinase enzyme. A control cell line was generated by transfecting an empty mock vector. All stable cell lines were selected for Hygromycin B resistance and consequently cultured in MEM + 10 % FCS + 1% Penicillin/Streptomycin + 50 μg/ml Hygromycin B (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNeasy Kit (Qiagen)
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
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| |
|
| Hybridization protocol |
Following fragmentation, cRNA were hybridized for 16 hr at 45C on Affymetrix Human Genome U133 Plus 2.0 Array in a Affymetrix Hybridization Oven 640. GeneChips were washed and stained in the Affymetrix Fluidics Station.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
RNA from cell culture with different NOD2 genotypes
|
| Data processing |
The data were analyzed with Gene Chip Operating Software 1.1. Represents the relative level of expression of a transcript. The median of the data is identified and each datum is weighted by how far it is from the median using One-step Tukey's Biweight Algorithm. A weighted mean is then calculated.
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| |
|
| Submission date |
Jun 29, 2010 |
| Last update date |
Jan 01, 2013 |
| Contact name |
Susanne Billmann |
| Organization name |
Institute of Clinical Molecular Biology Christian-Albrechts-Universität Kiel
|
| Street address |
Schittenhelmstraße 12
|
| City |
Kiel |
| ZIP/Postal code |
D-24105 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE22611 |
NOD2 and desease associated variant NOD2-L1007fsinsC dependent genomewide transcriptional regulation in stable Flp-In HEK cells |
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