|
| Status |
Public on Apr 09, 2012 |
| Title |
Wbp7-/-, untreated, replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
Wbp7-/-, untreated
|
| Organism |
Mus musculus |
| Characteristics |
strain background: mixed C57BL/6 (94%) and 129/Ola (6%)
genotype/variation: Wbp7-/-
cell type: Primary BMDM cells (7th day of differentiation)
treatment: No treatment
|
| Treatment protocol |
Macrophages were stimulated with lipopolysaccharide (LPS, 10 ug/ml)
|
| Growth protocol |
Bone marrow cells isolated from mixed C57BL/6 (94%) and 129/Ola (6%) mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% β-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNeasy from Qiagen with DNAse I treatment
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled cDNA targets were synthesized starting from 150 ng of totRNA. Double stranded cDNA synthesis and related cRNA amplification was performed with Ambion® WT Expression Kit (Ambion, Austin, TX). cRNA was reverse transcribed to cDNA fragmented and terminal labeled with Affymetrix GeneChip® WT Teminal Labeling Kit (Affymetrix, Santa Clara, CA). All steps of the protocol were performed according to manufacturer’s specifications. Efficacy of fragmentation procedure was checked on Agilent Bioanalyzer 2100.
|
| |
|
| Hybridization protocol |
GeneChip Hybridization, Wash and Stain Kit (Affymetrix cat #900720) is used to prepare the Hybridization Cocktail. The kit contains mix for target dilution, DMSO at a final concentration of 7% and pre-mixed biotin-labelled control oligo B2 and bioB, bioC, bioD and Cre controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM, respectively. These control oligos serve as positive controls for hybridization. Biotin-labeled sense targets were diluted in hybridization buffer at a concentration of 25 ng/ul and denatured at 99 °C for 5 minutes. Hybridization cocktails were incubated at 45 °C for 5 minutes and centrifuged at maximum speed for 1 minute prior to loading into the GeneChip array cartridge. Hybridizations were performed for 16 h at 45 °C in a rotisserie oven at 60 RPM. Upon hybridization, GeneChip cartridges were washed and stained with GeneChip Hybridization, Wash and Stain Kit in the Affymetrix Fluidics Station 450 following the FS450_0007 standard protocol. The cartridge is then loaded into the GeneChip Scanner 3000 7G.
|
| Scan protocol |
GeneChip arrays are scanned using default parameters. Affymetrix GeneChip Command Console software (AGCC) was used to acquire GeneChip images and generate .DAT and .CEL files.
|
| Description |
KO_UT_rep1
|
| Data processing |
CELs files were imported into Partek Genomics Suite software 6.5 (build 6.11.0321) and RMA normalized. Analysis was limited to probesets matching a known transcript.
|
| |
|
| Submission date |
Jul 27, 2011 |
| Last update date |
Apr 09, 2012 |
| Contact name |
Iros Barozzi |
| E-mail(s) |
iros.barozzi@meduniwien.ac.at
|
| Organization name |
Medical University Vienna
|
| Street address |
Borschkegasse 8a
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE30971 |
The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis. [Expression Profile] |
| GSE30973 |
The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis |
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