|
| Status |
Public on Oct 01, 2009 |
| Title |
C3Bir-Il10-/-_stimulated_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Bone marrow-derived macrophages, stimulated
|
| Organism |
Mus musculus |
| Characteristics |
strain: C3H/HeJBir-Il10tm1Cgn
gender: Male
cell type: Bone marrow-derived macrophages
protocol: stimulated with flagellin
|
| Growth protocol |
Cells were cultured in polystyrene 6-well culture plates (Corning, Corning, NY) at a density of 3 x 106/mL in RPMI1640 medium plus 10% fetal calf serum for six days. Culture medium was changed every third day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells from each donor in medium only and medium + flagellin (3 wells each) were harvested and pooled in a total of 2 mL Trizol (Invitrogen, Carlsbad, CA.). After passing the lysate through a 20G needle 5-6 times, RNA was extracted following the manufacturer’s protocol. RNA quality and yield was determined using the Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip assay (Agilent Technologies Inc, Palo Alto, CA).
|
| Label |
Biotin
|
| Label protocol |
Following reverse transcription with an oligo(dT)-T7 primer (Affymetrix, Santa Clara, CA), double-stranded cDNA was synthesized with the Superscript double-stranded cDNA synthesis custom kit (Invitrogen). The cDNA was linearly amplified and labeled with biotinylated nucleotides (Enzo Diagnostics, Farmingdale, NY) in an in vitro transcription reaction using T7 RNA polymerase.
|
| |
|
| Hybridization protocol |
Biotin-labeled and fragmented cRNA (15 µg) was hybridized onto MOE430v2.0 GeneChip™ arrays (Affymetrix) for 16 hours at 45°C. Post-hybridization staining and washing were performed according to manufacturer's protocols using the Fluidics Station 450 instrument (Affymetrix).
|
| Scan protocol |
The arrays were scanned with a GeneChip™ Scanner 3000 laser confocal slide scanner. The images were quantified using GeneChip(TM) Operating Software (GCOS) v1.2 and expression values summarized using the Robust MultiChip Average function in the R/affy package.
|
| Description |
Bone marrow-derived macrophages were cultured for six days. On the last day of culture, cells were stimulated with Cbir-flagellin (1 µg/mL culture medium) for 4 hours.
|
| Data processing |
An analysis of variance (ANOVA) model was applied and F1, F2, F3 and Fs test statistics were constructed along with their permutation p-values. False discovery rate (FDR) was then assessed using the R/qvalue package to estimate q-values from calculated test statistics.
|
| |
|
| Submission date |
Mar 20, 2009 |
| Last update date |
Mar 20, 2009 |
| Contact name |
Andre Bleich |
| E-mail(s) |
[email protected]
|
| Phone |
++49 511 532 8714
|
| Organization name |
Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School
|
| Street address |
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE15318 |
Cdcs1 a major colitis susceptibility locus in mice; subcongenic analysis reveals genetic complexity |
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