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Status |
Public on Jan 23, 2024 |
Title |
Tconv_CD28_Donor4 |
Sample type |
SRA |
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Source name |
Peripheral blood
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Organism |
Homo sapiens |
Characteristics |
tissue: Peripheral blood cell type: Conventional CD4 T cell treatment: IL-2 and anti-CD3/CD28 agonistic mAbs
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Growth protocol |
Naïve Tconv and Treg cells were sorted from buffy coats from healthy donors (Sanquin) by flow cytometry. After cell sorting (day 0), naïve Tconv and tTreg cells were cultured for 7 days at 37°C/5% CO2 in 96-well round bottom plates (Corning, Thermo Scientific; 1×10^4 cells per well) in 100 µl IMDM (Capricorn Scientific) with 8% FCS (Serana), penicillin/streptomycin (Gibco) and IL-2 (300 IU/ml, DuPont Medical). Soluble agonistic mAb to CD3 (0.1 µg/ml, clone CLB-T3/4.E, IgE isotype, Sanquin) was used for TCR/CD3 stimulation, and soluble agonistic mAb to either CD28 (0.2 µg/ml, clone CLB-CD28/1, Sanquin) or TNFR2 (2.5 µg/ml, clone MR2-1, Hycult Biotech) for costimulation. On day 4, 100 µl supplemented IMDM including IL-2 (300 IU/ml) was added to the culture. On day 7, cells were collected for transcriptomic analysis.
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Extracted molecule |
total RNA |
Extraction protocol |
T cells were washed in cold PBS and resuspended in RLT buffer (Qiagen). Total RNA isolation was performed according to the 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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
6377_13_47_Donor4_Tconv_aCD28_AGTCAAC_S86
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Data processing |
The 65 bp single end reads were mapped to the human reference genome (hg38, genome_snp_tran) using HISAT2 (version 2.1.0). Read counts were generated using Gensum (https://github.com/NKI-GCF/gensum). Ensembl GTF version 102 was used as a reference. BAM files were imported in Qlucore Omics Explorer software (version 3.8) with GRCh38.104.gtf as reference genome, using trimmed mean of M values (TMM) normalization. !Sample_data_processing = Transcripts with less than 10 reads in at least 80% of the samples were excluded from downstream expression analysis, as well as remaining transcripts with low expression in all samples. Assembly: GRCh38 Supplementary files format and content: Tab-delimited text file including data after TMM normalization and log-transformation
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Submission date |
Jan 18, 2024 |
Last update date |
Jan 23, 2024 |
Contact name |
Mark Mensink |
E-mail(s) |
m.mensink@lumc.nl
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Organization name |
Leiden University Medical Center
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Department |
Immunology
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Street address |
Albinusdreef 2
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City |
Leiden |
ZIP/Postal code |
2333ZA |
Country |
Netherlands |
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Platform ID |
GPL16791 |
Series (1) |
GSE253540 |
Treg cells from human blood differentiate into non-lymphoid tissue-resident effector cells upon TNFR2 costimulation |
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Relations |
BioSample |
SAMN39484697 |
SRA |
SRX23274524 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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