|
| Status |
Public on Oct 27, 2014 |
| Title |
OP9_GFP_donor4 |
| Sample type |
RNA |
| |
|
| Source name |
CD34+, OP9-GFP, donor4
|
| Organism |
Homo sapiens |
| Characteristics |
co-culture: OP9-GFP
|
| Treatment protocol |
CD34+ thymocytes were purified out of pediatric thymus samples using MACS and seeded onto confluent OP9-GFP (control) or OP9-DLL1 plates for 48h in α-MEM media supplemented with 20% heat-inactivated FCS plus 100U penicilin, 100 µg/ml streptomycin, 2 mM L-glutamine and cytokines SCF, Flt-3 and IL-7 at 5 ng/ml each.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested with the miRNeasy mini kit (Qiagen) with Dnase treatment on-column.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Low Input Quick Amp Labling kit (Agilent) according to the manufacturer's instructions, followed by miRNeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity > 6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x GEx Hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to the array for 17 hours at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner with SureScan High-Resolution Technology using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3µm, Dye channel is set to Green).
|
| Description |
Gene expression of CD34+ cells after a 48h coculture with an OP9-GFP feeder layer.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters (protocol GE1-105_Dec08 and Grid: 041648_D_F_20120615) to obtain background subtracted and spatially detrended Processed Signal intensities. Normalization was performed using the VSN-package (Bioconductor) and background correction was performed during the normalization process, selecting those probes detecting a 2 times higher expression than the average of the DarkCorner probes in at least 60% of the samples one treatment.
|
| |
|
| Submission date |
Sep 29, 2014 |
| Last update date |
Oct 28, 2014 |
| Contact name |
Annelynn Wallaert |
| E-mail(s) |
[email protected]
|
| Organization name |
UGent
|
| Street address |
De Pintelaan 185
|
| City |
Gent |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL19197 |
| Series (2) |
| GSE61871 |
Development of gene expression signatures with lncRNAs for coculture of CD34+ T-cells with an OP9-DLL1 feeder layer |
| GSE62006 |
The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia |
|
