|
| Status |
Public on Jan 30, 2023 |
| Title |
AA_CD8_Tofa_300nM_D3_KT_19 |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood CD8+ T-cells, Tofacitinib
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: healthy
cell type: CD8+ T-cells
treatment: Tofacitinib
concentration: 300nM
timepoint: 40hrs
donor: D3
|
| Treatment protocol |
CD8+ T-cells were stimulated in vitro with plate bound anti-CD3 (OKT3) in complete media in the presence or absence of different JAK inhibitors; tofacitinib, ruxolitinib and a selective JAK1 inhibitor at 300 nM. Soluble anti-CD28 were added to the CD8+ cultures to facilitate co-stimulation. After 40h, supernatants were harvested for mediator analysis and the cells were washed and resuspended in RLT buffer (Qiagen) for RNA isolation.
|
| Growth protocol |
Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Sepmate technique (Stemcell Technologies) from heparinized venous blood from healthy volunteers. Primary human CD8+ T-cells were further isolated using a negative selection kit according to protocol (Miltenyi Biotech).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Mini kit with on-column DNA digestion (QIAGEN, Hilden, Germany) and eluted in 50 ul RNase-free water.
RNA was diluted to 5ng/ul and used as input to create RNA libraries using TruSeq Stranded mRNA kit (Illumina, CA, USA) following standard instructions. Libraries were validated on the BioAnalyzer (Agilent, CA, USA) using DNA1000 chip and the concentration was determined using Quant-iT dsDNA High Sensitivity assay kit on the Qubit fluorometer (Thermo Fisher, MA, USA). Sample libraries were pooled in equimolar concentrations and diluted and denatured according to Illumina guidelines. The pool was then sequenced using a High Output 2 x 76 bp kit on an Illumina NextSeq500.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
AA_CD8_Tofa_300nM_D3_KT_19_S11
PolyA RNA
Processed data file: combined.counts.txt
Processed data file: combined.gene.sf.tpm.txt
|
| Data processing |
Base calling was done by bcl2fastq from Illumina.
Reads were mapped with HiSat2 (2.0.4).
Counts were generated with featurecounts (1.4.4).
TPMs were generated using Sailfish (0.10.1).
All processing was wrapped together with bcbio 0.9.9.
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: combined.counts.txt: Tab-delimited text file includes counts.
Supplementary_files_format_and_content: combined.gene.sf.tpm.txt: Tab-delimited text file includes TPMs.
|
| |
|
| Submission date |
Jan 27, 2017 |
| Last update date |
Jan 30, 2023 |
| Contact name |
Elisabeth Israelsson |
| E-mail(s) |
elisabeth.israelsson@astrazeneca.com
|
| Organization name |
AstraZeneca, IMED RIA
|
| Department |
Translational Biology
|
| Street address |
Pepparedsleden 1
|
| City |
Molndal |
| ZIP/Postal code |
SE-431 83 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE94235 |
Selective Janus kinase 1 inhibition resolves inflammation and restores hair growth offering a viable treatment option for alopecia areata |
| GSE94237 |
JAK inhibition in human CD8+ T-cells and C3H/HeJ Mice with Alopecia Areata In Vivo |
|
| Relations |
| BioSample |
SAMN06277343 |
| SRA |
SRX2524196 |
