|
| Status |
Public on Jul 10, 2020 |
| Title |
Tconv_CD3-CD28_Donor2 |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Conventional CD4+ T cell
treatment: IL-2 and anti-CD3/CD28 agonistic mAbs
individual: Donor2
|
| Treatment protocol |
Following the expansion protocol, 1x10^5 T cells were either unstimulated or restimulated using anti-CD3/CD28 mAbs for 24 hours, in the presence of IL-2, and were subsequently harvested for whole transcriptome analysis.
|
| Growth protocol |
Naïve Tconv and Treg cells were sorted from human buffy coats from male healthy donors (Sanquin) by flow cytometry. Human sorted Tconv and Treg cells were plated in 96-well round bottom plates (Greiner; 1x10^4 cells per well) and cultured in IMDM (Gibco, Life Technologies), supplemented with 8% FCS (Sigma), penicillin/streptomycin (Roche) and 300 IU/mL IL-2 (DuPont Medical) (T cell medium) at 37 °C/5% CO2. Activating mAbs to CD3 (clone CLB-T3/4.E, Sanquin, 0.1 µg/ml) and CD28 (clone CLB-CD28/1, Sanquin, 0.2 µg/ml,) were used for T cell expansion. From day 7 to 14, T cells (5x10^5 cells/mL) were plated in 24-well plates (Greiner). After 1 or 2 weeks of culture, Tconv and Treg cells (1 x 10^6 cells/mL) were cultured in 6-well plates (Corning) for 4 days in fresh T cell medium supplemented with 300 IU/mL IL-2, but without anti-CD3/CD28 mAbs. Dead cells were removed by Ficoll density gradient centrifugation prior to restimulation experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Restimulated T cells were washed in cold PBS and resuspended in RLT buffer (Qiagen). Total RNA isolation was performed according to manufacturer’s protocol using the RNeasy MinElute Cleanup Kit (Qiagen) including an on-column DNAse digestion (Qiagen). Quality and quantity of the total RNA were assessed on a 2100 Bioanalyzer using a Nano chip (Agilent).
Library preparation was performed on RNA samples with a measured RNA Integrity Number (RIN) between 8.0 and 10.0. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc.) according to manufacturer’s instructions (Illumina, Part # 15031047 Rev. E). RNA libraries were prepared for sequencing using standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Sample 6: 4848_06_56_Tconv_stim_24h_GPA33_D6_GTTTCGG
|
| Data processing |
The 65 bp single end reads were mapped to the human reference genome (hg38) using TopHat (version 2.1.0).
TopHat was supplied with a known set of gene models based on Ensembl GTF version 77 and was guided to use the first-strand as the library-type. As additional parameters --prefilter-multihits and –no coverage were used.
Genecounts were generated using a custom script that gives the same output as HTSeq-count.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-separated file with number of reads per gene/sample.
|
| |
|
| Submission date |
Oct 08, 2019 |
| Last update date |
Jul 10, 2020 |
| Contact name |
Mark Mensink |
| E-mail(s) |
m.mensink@lumc.nl
|
| Organization name |
Leiden University Medical Center
|
| Department |
Immunology
|
| Street address |
Albinusdreef 2
|
| City |
Leiden |
| ZIP/Postal code |
2333ZA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE138603 |
Response of expanded human conventional and regulatory T cells to CD3/CD28-mediated activation [Exp1] |
| GSE138605 |
Response of expanded human conventional and regulatory T cells to CD3/CD28-mediated activation (Exp1) and to CD28 or TNFR2 costimulation (Exp2) |
|
| Relations |
| BioSample |
SAMN12995254 |
| SRA |
SRX6966147 |
