|
| Status |
Public on May 21, 2020 |
| Title |
veh_ac_3 |
| Sample type |
SRA |
| |
|
| Source name |
CD8+ T cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
stimulation: Acute
treatment: vehicle
|
| Treatment protocol |
Following 48 h of initial stimulation, T-cells were cultured at 1 million cells per mL in in RPMI-1640 media containing 10% FBS, 2 mM L-glutamine, 5 mM beta-mercaptoethanol, and 10 ng/mL IL-2. and with (chronic) or without (acute) plate-bound anti-CD3 (3 mg/mL). This was repeated every 48 h until eight days following initial stimulation, at which point cells were harvested for RNA. Where indicated, N-acetylcysteine was added at 10 mM beginning 48 h after initial stimulation. For OT-I cells, T-cells were co-cultured with B16-F10 melanoma cells that had been treated with 1 ng/mL IFN-g to induce MHC-I expression. Cells were co-cultured at 1 million T-cells per mL with (chronic) or without (acute) the addition of 1 mM SIINFEKL. This was repeated every 48 h until eight days following initial stimulation, at which point cells were harvested for RNA.
|
| Growth protocol |
Polyclonal T-cells were isolated from mouse spleen and inguinal lymph nodes of using the Dynabeads Untouched CD8+ T-cell kit. T-cells were activated with platebound anti-CD3 (2C11, 3 mg/mL) and anti-CD28 (19.51, 1 mg/mL) for 48 h in RPMI-1640 media containing 10% FBS, 2 mM L-glutamine, 50 mM beta-mercaptoethanol, and 10 ng/mL IL-2. For activation of OT-I transgenic CD8+ T-cells, cells were cultured at 10 million cells per mL in the presence of 1 mM SIINFEKL peptide.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from cells using Trizol reagent
Libraries were prepared from poly-A+ RNA with the NEBNext Ultra RNA library prep kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.
Adapter sequences were trimmed and validated read pairs were aligned to the mouse genome with TopHat2.1.1 with default parameters.
Uniquely aligned reads were used to count gene features with 'htseq-count'.
Normalization and differential expression was determined using the 'edgeR' package.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text of normalized RPKM values per gene
|
| |
|
| Submission date |
Oct 04, 2019 |
| Last update date |
Sep 22, 2020 |
| Contact name |
Bryan King |
| E-mail(s) |
kingb2@mskcc.org
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Department |
Cancer Biology and Genetics
|
| Lab |
Craig Thompson Lab
|
| Street address |
430 E 67th St RRL-469
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE138459 |
Reversible mitochondrial dysfunction drives terminal T-cell exhaustion |
|
| Relations |
| BioSample |
SAMN12915997 |
| SRA |
SRX6952579 |
