|
| Status |
Public on Nov 05, 2023 |
| Title |
11_SIINFEKL |
| Sample type |
SRA |
| |
|
| Source name |
lymphoid organs (lymph nodes and spleen)
|
| Organism |
Mus musculus |
| Characteristics |
tissue: lymphoid organs (lymph nodes and spleen)
cell type: FACS: Live/Dead -, CD45.1+, CD8a+ (OT-I T cells)
time: 24h
treatment: SIINFEKL
|
| Treatment protocol |
Cells were cultured in conditions of 2 hours no stimulation, 2 hours stimulation with 100ng/mL TGF-β, 24 hours stimulation with 100nM N4 (SIINFEKL), and 24 hours stimulation with 100nM N4 (SIINFEKL) and 100ng/mL TGF-β.
|
| Growth protocol |
To enrich bulk T cells from single cell suspensions, we used a mouse-specific T cell negative isolation MACS (STEMCELL Technologies, Canada). We plated 0.5–1 × 106 T cells per well in 96-well V-bottom tissue culture plates. We cultured cells in mouse RP10 media (RPMI 1640 supplemented with 10% FBS, 2mM L-glutamine, 100 U/mL penicillin-streptomycin, 1mM sodium pyruvate, 0.05mM β mercaptoethanol, and 1mM HEPES) at 37°C with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
None. 500 OT-I T cells were sorted directly into the lysis buffer of the SMART-Seq v4 Ultra Low Input RNA Kit (Takara).
RNAseq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) per the manufacturer's guidelines.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
low input bulk RNAseq
|
| Data processing |
Base calls were processed to FASTQs on BaseSpace (Illumina), and a base call quality-trimming step was applied to remove low-confidence base calls from the ends of reads.
Reads were processed using workflows managed on the Galaxy platform. Reads were trimmed by 1 base at the 3′ end then trimmed from both ends until base calls had a minimum quality score of at least 30. Any remaining adapter sequence was removed as well.
To align the trimmed reads, STAR aligner (v2.4.2a) was used with the GRCm38 reference genome.
Gene counts were generated using HTSeq-count (v0.4.1).
Assembly: GRCm38
Supplementary files format and content: comma-separated values file includes raw counts for each Sample
|
| |
|
| Submission date |
Nov 02, 2023 |
| Last update date |
Nov 05, 2023 |
| Contact name |
Martin Prlic |
| E-mail(s) |
mprlic@fredhutch.org
|
| Phone |
2066672216
|
| Organization name |
Fred Hutchinson Cancer Center
|
| Lab |
Prlic
|
| Street address |
1100 Fairview Ave, Mail stop E5-110
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98102 |
| Country |
USA |
| |
|
| Platform ID |
GPL30172 |
| Series (2) |
| GSE246932 |
TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner (RNA-Seq) |
| GSE246933 |
TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner |
|
| Relations |
| BioSample |
SAMN38083857 |
| SRA |
SRX22361166 |
