|
| Status |
Public on Jun 08, 2012 |
| Title |
NOD B cells, 2h anti-IgM stimulation, biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Splenic NOD B cells, 2h culture with anti-IgM stimulation
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: NOD/ShiLtJ (NOD)
age: 6 weeks
cell type: splenic B cells
stimulation: anti-IgM stimulated
|
| Treatment protocol |
Purified B cells from each lot were resuspended at 1x10<7> cells/ml for 2h in complete RPMI media alone or with 10µg/ml AffiniPure goat anti-mouse IgM F(ab’)2 fragments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA from cultured cells was performed according to the manufacturer's instructions and quality was assessed using an Agilent 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (Agilent Technologies, Palo Alto, CA).
|
| Label |
biotin
|
| Label protocol |
Following reverse transcription with an Oligo(dT)-T7 primer (Affymetrix, Santa Clara, Ca), double stranded stranded cDNA was synthesized with the GeneChip® Expression 3’-Amplification One-cycle kit (Affymetrix, Santa Clara, Ca). In an in vitro (IVT) reaction with T7 RNA polymerase, the cDNA was linearly amplified and labeled with biotinylated nucleotides (Affymetrix).
|
| |
|
| Hybridization protocol |
10ug of fragmented RNA was hybridised for 16h at 45C on Affymetrix Mouse Genome 4.30 2.0 Arrays. Washing and staining performed as per manufacturer's instruction on Affymetrix Fludics Station 450 instrument.
|
| Scan protocol |
Arrays were scanned with a GeneChip Scanner 3000 laser confocal slide scanner (Affymetrix).
|
| Description |
Gene expression data from splenic NOD B cells cultured for 2h with anti-IgM stimulation
|
| Data processing |
Analysis of data was performed using GeneSpring 12.0 software (Agilent). Samples were preprocessed with RMA. All samples were normalised on a per gene basis to the median of total samples. The data was filtered for genes which had a raw expression level of >50 in at least one sample.
|
| |
|
| Submission date |
Apr 16, 2012 |
| Last update date |
Jun 08, 2012 |
| Contact name |
Pablo Alejandro Silveira |
| E-mail(s) |
P.Silveira@garvan.org.au
|
| Phone |
+61-2-9295 8456
|
| Fax |
+61-2-9295 8404
|
| Organization name |
Garvan Institute
|
| Department |
Immunology Program
|
| Street address |
384 Victoria St
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE37294 |
Comparison of gene expression in NOD versus NOD.NOR-Chr4 (NR4) splenic B cells. |
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