|
| Status |
Public on Aug 24, 2012 |
| Title |
mouse thymus wt DN3 8 |
| Sample type |
RNA |
| |
|
| Source name |
wild-type mouse thymus
|
| Organism |
Mus musculus |
| Characteristics |
sample group: DN3 +/-
genotype: +/-, Foxn1 heterozygous
cell type: DN3, CD4/CD8-negative stage 3 thymocytes
|
| Treatment protocol |
Cells were directly lysed for RNA isolation
|
| Growth protocol |
Cells were directly isolated from indicated tissue sources by flow cytometry
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cell samples were lysed using SuperAmp™ Lysis Buffer following manufacturer’s instructions and stored at -80°C. DNA microarray analysis of gene expression was performed at Miltenyi Biotec´s Genomics Service facility (Bergisch Gladbach, Germany), using Agilent technology (Agilent Technologies, Inc.). SuperAmplification was performed according to manufacturer’s instruction. Briefly, cell samples were collected in 6.4 μl lysis buffer (including detergent, tRNA and Protease), the mRNA extracted using magnetic beads and transcribed into cDNA using tagged random and oligo(dT) primer. First strand cDNA was 5’ tagged using terminal deoxynucleotidyl transferase (Fermentas). Tagged cDNA was globally amplified (Expand Long Template PCR System DNA Pol Mix, Roche) using primers complementary to the tag sequence. Amplified cDNAs were quantified using ND-1000 Spectrophotometer (NanoDrop Technologies, Inc). Integrity of the cDNA was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies, Inc.)
|
| Label |
Cy3
|
| Label protocol |
250 ng of the purified PCR product (NucleoSpin® Extract II Kit, Macherey & Nagel) was labeled with Cy3-dCTP (GE Healthcare) in a Klenow Fragment reaction.
|
| |
|
| Hybridization protocol |
0.5 μg Cy3 labeled and purified (CyScribe GFX Purification Kit, GE Healthcare) cDNAs in hybridization buffer were hybridized overnight (17 hours, 65°C) to 8x60K Agilent Whole Mouse Genome Oligo Microarrays using Agilent’s recommended hybridization chamber and oven. Following hybridization, the microarrays were washed once with Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated (37°C) Agilent Gene Expression Wash Buffer 2 containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES, v.10.7.3.1) was used to read out and process the microarray image files.
|
| Description |
gene expression analysis of SuperAmplified™ polyA RNA
|
| Data processing |
FES derived output data files were further analyzed using GeneSpring GX (v11.5.1).
|
| |
|
| Submission date |
Oct 26, 2011 |
| Last update date |
Aug 24, 2012 |
| Contact name |
Stefan Tomiuk |
| E-mail(s) |
stefant@miltenyibiotec.de
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
Bioinformatics
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch-Gladbach |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE33268 |
Synergistic, context-dependent and hierarchical functions of hematopoietic progenitor niche components. |
|
