|
| Status |
Public on Jun 12, 2017 |
| Title |
MYB x_17 |
| Sample type |
RNA |
| |
|
| Source name |
CD4+ T cells polarized towards Th2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
subject: 7
|
| Treatment protocol |
Human Naïve CD4+ T cells were isolated from fresh buffy coats with Miltenyis Naïve CD4+ T Cell Isolation Kit II, according to the manufacturer’s instructions. Cells were either transfected in a cuvette with 600nM human on target plus SMART pool against MAF, MYB and GATA3 (ThermoFisherScientific Inc, USA), non-targeting siRNA (ThermoFisherScientific Inc) or with transfection buffer using the Amaxa transfection program U-014. Five hours after the nucleofection cells were washed, activated and polarized towards TH2 for or 24h. The cells were activated with platebound anti-CD3 (500 ng/ml), 500 ng/ml soluble anti-CD28, 5 µg/ml anti-IL-12, 10 ng/ml IL-4 and 17 ng/ml IL-2 (R&D Systems).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For microarray experiments the cells were harvested at 24h of polarization and lysed in 600 ll Qiazol.
|
| Label |
Cy3
|
| Label protocol |
The expression profiling 200 ng of total RNA from each sample was labeled with Cy3 and transcribed to cRNA using the Agilent Quick Amp Labeling Kit, one color. The labeled cRNA was purified with the RNEasy Mini Kit from Qiagen. The labeling efficiency was assessed using the NanoDrop ND-1000 UV-Vis Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 µg Cy3 labeled cRNA from each sample was hybridized to Agilent SurePrint G3 Human GE v2 8x60K slides. The microarray slides were hybridized for 17 hours at 65°C in a rotating oven.
|
| Scan protocol |
After washing according to the manufacturer’s instructions, the slides were analyzed using the Agilent Microarray scanner 2505C with default settings for all parameters.
|
| Data processing |
Gene expression data was obtained with Agilent`s feature extraction software (version 10.7.3.1), and next quantile normalized
|
| |
|
| Submission date |
Feb 28, 2017 |
| Last update date |
Jun 12, 2017 |
| Contact name |
Danuta Gawel |
| E-mail(s) |
danuta.gawel@liu.se
|
| Organization name |
Linköping University
|
| Street address |
Garnisonsvägen
|
| City |
Linköping |
| ZIP/Postal code |
58185 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE60683 |
LASSIM -a network inference toolbox for genome-wide mechanistic modeling |
| GSE65880 |
LASSIM -a network inference toolbox for genome-wide mechanistic modeling [MAF, MYB & GATA3 siRNA] |
|
