|
| Status |
Public on Sep 01, 2009 |
| Title |
si-Eos Treg |
| Sample type |
RNA |
| |
|
| Source name |
si-Eos GFP + CD4+CD25+ T cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: natural Tregs
transfection protocol: Eos shRNA knockdown
strain: Balb/C
secondary lymphoid tissue: Spleen and Lymph nodes
|
| Treatment protocol |
Natural Tregs enriched for CD25+ using Miltenyi’s magnetically labeled beads, transduced with lentivirus containined Eos or Renilla luciferase gene specific shRNAs, and incubated for 60 hrs in Human IL-2 supplemented complete RPMI. GFP+ cells were sorted out for Gene Expression analysis using the FACSAria I.
|
| Growth protocol |
CD4 T cells were isolated from Balb/C mice and enriched for CD25+ and CD25- populations using Miltenyi’s magnetically labeled beads before sorting. Cells were maintained in Complete RPMI 1640.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sorted cells were pelleted suspended in Trizol and stored at -80ºC prior to RNA extraction using the Trizol reagent according to the manufacturer's instructions. RNA integrity was analyzed using an Agilent 2100 Bioanalyzer, the RNA 6000 Pico, and Nano Kits (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Standard labeling using Cyanine-3 Labeled CTPs was carried out after cDNA was synthesized using the Low RNA input Fluorescent linear Amplification kit ( Agilent Technologies) according to the manufacturers protocol.
|
| |
|
| Hybridization protocol |
Samples were hybridized to separate Agilent 4x 44K whole mouse genome microarray chips at 65ºC for 17hrs
|
| Scan protocol |
Expression array Chips were scanned using Agilent Scanner controlled by Agilent Scan Control 7.0 Software and data was extracted using Agilent Feature Extraction 9.5.3.1 software.
|
| Description |
Gene Expression of si-Eos Natural Treg
|
| Data processing |
Agilent FE processed signal intensities were imported into Genespring GX 9.0.1 (Agilent Technologies).
Log2 transformation and Cross-array quartile normalization was carried out on all intensities higher than 5.
|
| |
|
| Submission date |
Jul 17, 2009 |
| Last update date |
Sep 01, 2009 |
| Contact name |
Fan Pan |
| E-mail(s) |
fpan1@jhmi.edu
|
| Phone |
410-6149130
|
| Fax |
410-6140549
|
| Organization name |
Johns Hopkins Medical Institions, School of Medicine
|
| Department |
Oncology
|
| Lab |
Dr. Drew Pardoll
|
| Street address |
1650 Orleans St CRB I Room 424
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE17166 |
Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells |
|
