|
| Status |
Public on Mar 07, 2013 |
| Title |
Tgfb+Il6_60hr_HJ |
| Sample type |
RNA |
| |
|
| Source name |
Th17 cells
|
| Organism |
Mus musculus |
| Characteristics |
time (hr): 60
treatment: Tgfb+Il6
cell type: Th17
|
| Treatment protocol |
For Th17 differentiation: 2ng/mL rhTGF-β1 (Miltenyi Biotec), 25ng/mL rmIl-6 (Miltenyi Biotec), 20ng/ml rmIl-23 (Miltenyi Biotec), and 20ng/ml rmIL-β1 (Miltenyi Biotec). Cells were cultured for 48 hours.
|
| Growth protocol |
Cd4+ T cells were purified from spleen and lymph nodes using anti-CD4 microbeads (Miltenyi Biotech) then stained in PBS with 1% FCS for 20 min at room temperature with anti-Cd4-PerCP, anti-Cd62l-APC, and anti-Cd44-PE antibodies (all Biolegend, CA). Naïve Cd4+ Cd62lhigh Cd44low T cells were sorted using the BD FACSAria cell sorter. Sorted cells were activated with plate bound anti-Cd3 (2μg/ml) and anti-Cd28 (2μg/ml) in the presence of cytokines.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA was performed according to the RNeasy Mini Kit (Qiagen) manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA (3' IVT Express Kit, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 12.5 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix microarray HT_MG-430A. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G
|
| Data processing |
Expression data was preprocessed using the RMA algorithm followed by quantile normalization, with the parameters in the ExpressionFileCreator module of the GenePattern suite set to default
|
| |
|
| Submission date |
Jan 31, 2013 |
| Last update date |
Mar 07, 2013 |
| Contact name |
Nir Yosef |
| E-mail(s) |
niryosef@berkeley.edu
|
| Phone |
(617) 714-7734
|
| Organization name |
UC Berkeley
|
| Department |
EECS
|
| Lab |
Yosef
|
| Street address |
Berkeley
|
| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (2) |
| GSE43955 |
Reconstruction of the dynamic regulatory network that controls Th17 cell differentiation by systematic perturbation in primary cells (Th17 differentiation timecourse) |
| GSE43970 |
Reconstruction of the dynamic regulatory network that controls Th17 cell differentiation by systematic perturbation in primary cells |
|
