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Sample GSM8022812 Query DataSets for GSM8022812
Status Public on Jan 23, 2024
Title Tconv_CD28_Donor4
Sample type SRA
 
Source name Peripheral blood
Organism Homo sapiens
Characteristics tissue: Peripheral blood
cell type: Conventional CD4 T cell
treatment: IL-2 and anti-CD3/CD28 agonistic mAbs
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description 6377_13_47_Donor4_Tconv_aCD28_AGTCAAC_S86
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
 
Submission date Jan 18, 2024
Last update date Jan 23, 2024
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 (1)
GSE253540 Treg cells from human blood differentiate into non-lymphoid tissue-resident effector cells upon TNFR2 costimulation
Relations
BioSample SAMN39484697
SRA SRX23274524

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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