|
| Status |
Public on Jan 27, 2013 |
| Title |
CD4+ D011.10+ CD44 high cells from sOVA immunized sOVA host, 5 days following transfer of D011.10+ T cells rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
in vivo activated mouse CD4 T cells
|
| Organism |
Mus musculus |
| Characteristics |
tissue: spleen/lymph nodes
strain: Balb/c
age: 6-8 weeks
|
| Treatment protocol |
LNs and spleens were pooled from DO11 Rag2-/- mice and CD4+ cells purified using magnetic beads. DO11 Rag2-/- cells were used for the transfers but DO11 Rag2+/+ cells were used for the naive group. 2-5 x 10^5 CD4+ cells were then intravenously injected recipients. Donor T cells were transferred into sOVA or WT Balb/c mice and, 24 hours later, these were immunized (IV) with 1-5 x 10^5 bone marrow-derived dendritic cells that were pre-activated with LPS and loaded with Ova peptide. Naïve or activated D011.10 CD4 T cells were isolated from pooled mouse spleen and lymph nodes using FACS sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol reagent and standard protocol.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was labeled with Cy3-CTP using the miRCURY LNA microRNA power labeling kit (Exiqon, Inc, Woburn, MA), according to manufacturers protocol
|
| |
|
| Hybridization protocol |
Cy3-labelled RNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent UCSF custom 8x15K miRNA arrays for 17 hours at 65°C in a rotating Agilent 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned on the Agilent scanner immediately after washing.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.6 (Agilent) using default parameters)
|
| |
|
| Submission date |
Mar 19, 2012 |
| Last update date |
May 03, 2013 |
| Contact name |
Andrea J Barczak |
| Organization name |
UCSF Sandler Center Functional Genomics Core Facility
|
| Street address |
1550 4th St Rock Hall 545
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL14557 |
| Series (1) |
| GSE36607 |
miRNA expression during in vitro mouse T cell activation (D011.10+ CD4 T cells) |
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