|
| Status |
Public on Jul 19, 2014 |
| Title |
CCd27 Post |
| Sample type |
RNA |
| |
|
| Source name |
coculture CD27+CD1a- TCRαβ-transduced, expanded
|
| Organism |
Homo sapiens |
| Characteristics |
developmental stage: child
culture type: in vitro culture from thymus stem cells
cell type: CD27+CD1a- TCRαβ-transduced, expanded
|
| Biomaterial provider |
UZ Ghent, according to protocol approved by ethical commission
|
| Treatment protocol |
No treatment applied.
|
| Growth protocol |
Cultured cells were cultured in alphaMEM supplemented with SCF, flt3 and interleukin-7.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
miRNeasy kit (Qiagen), following manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
RNA concentration was determined with the Qubit RNA Assay using the Qubit 2.0 Fluorometer (Life Technologies), RNA purity was determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit.
|
| |
|
| Hybridization protocol |
A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip Human Gene 2.0 ST Arrays followed by staining and washing in a GeneChip® fluidics station 450 (Affymetrix) according to the manufacturer’s procedures.
|
| Scan protocol |
To assess the raw probe signal intensities, chips were scanned using a GeneChip® scanner 3000 (Affymetrix).
|
| Data processing |
Data were processed with RMA as implemented in the XPS package of Bioconductor. Reported values are on log2-scale.
|
| |
|
| Submission date |
Jul 19, 2013 |
| Last update date |
Jul 19, 2014 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL16686 |
| Series (1) |
| GSE49039 |
Comparison of gene expression from thymocyte populations and equivalent OP9-DL1 cultured cells |
|
