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Sample GSM8022802

Query DataSets for GSM8022802

Status

Public on Jan 23, 2024

Title

tTreg_CD28_Donor1

Sample type

SRA

Source name

Peripheral blood

Organism

Homo sapiens

Characteristics

tissue: Peripheral blood
cell type: Regulatory 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_3_33_Donor1_tTreg_aCD28_TTAGGCA_S84

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