|
| Status |
Public on Oct 01, 2015 |
| Title |
Spleen_2 |
| Sample type |
RNA |
| |
|
| Source name |
Spleen M195-specific CD8+ T cells sorted from spleens of HMPV-infected mice at day 7 post-infection.
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL6/J
gender: female
age: 8-12 weeks
genotype/variation: B6-Kb0Db0,B7.2 transgenic (B7tg) mice
infected with: human metapneumovirus (HMPV)
tissue: spleen
cell type: Spleen CD8+ T cells
|
| Treatment protocol |
sort protocol: HMPV (pathogenic clinical strain TN/94-49, genotype A2) was grown and titered in LLC-MK2 cells. Mice were anesthetized with ketamine-xylazine and infected intranasally with 1x106 PFU of HMPV. Lymphocytes were isolated from spleens and lungs of infected animals. To obtain sufficient quantities of primary epitope-specific CD8+ T cells, on day 7 after HMPV infection the spleens and lungs from three mice were pooled together after processing. Samples were stained, sorted, and RNA purified on separate days in independent experiments. Cells were processed in ice-cold R10 media containing 10nM dasatinib to prevent activation and TCR signaling. Splenocytes were depleted of B cells by incubation on goat anti-mouse IgG and IgM coated (100μg/mL) T-75 flasks at 106 cells/mL for 1hr at 37C. Both lung and spleen cells were stained for viability, CD19, CD8, and M195-tetramers (both PE- and APC-conjugated). Surface/tetramer staining was performed for 1 hour at RT in PBS containing 1% FBS and 50nM dasatinib. Splenocytes were also stained for CD44 and CD62L to obtain control naïve cells. Dual-tetramer positive CD8+ T cells were sorted using a BD FACSAria III. Samples were maintained at 4C for the entirety of the sort and purity was 97–99% for all populations.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from sorted naive, spleen, lung, and secondary lung M195-specific CD8+ T cells using an RNeasy kit (QIAGEN) according to the manufacturer's instructions. On-column DNase digestion was performed and eluted RNA was quantified and checked for integrity using an Agilent 2100 Bioanalyzer. RNA was judged as suitable only if samples showed intact bands of 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RNA integrity number (RIN) above 8.0.
|
| Label |
Biotin
|
| Label protocol |
The FL-Ovation cDNA Biotin Molecule V2 (NuGen) was used to fragment and label single-stranded cDNA targets for subsequent hybridization.
|
| |
|
| Hybridization protocol |
The Affymetrix GeneChip Mouse Gene 1.1 ST 16-array plate consists of 16 single Gene 1.1 ST arrays arranged into the standard 96 well plate format. Array hybridization, washing and scanning were performed on a GeneTitan Instrument according to the manufacturer’s recommendations.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneTitan instrument.
|
| Description |
Spleen M195-specific CD8+ T cells replicate #2
|
| Data processing |
Microarray data were processed using the oligo package implementation of rma in R software.
|
| |
|
| Submission date |
Dec 16, 2013 |
| Last update date |
Oct 01, 2015 |
| Contact name |
Pengcheng Lu |
| E-mail(s) |
pengcheng.lu@vanderbilt.edu
|
| Organization name |
Vanderbilt University
|
| Street address |
2200 Pierce Avenue
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL11533 |
| Series (1) |
| GSE53349 |
CD8+ T cells during acute viral respiratory infection are uniquely differentiated and regulated by multiple inhibitory receptors |
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