characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-12-15T17:02:34Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-04-05T15:13:54Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA103958003
characteristics.characteristic: External Id
characteristics.text: Radboud University Medical Center (Radboudumc)
characteristics.characteristic: INSDC center name
characteristics.text: 2017-12-15T17:02:34Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-04-05T15:13:54Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1647171
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5622:rhTNF_alpha treatment_Donor 4
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: CD4-positive, alpha-beta memory T cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000897
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: CD4+ T cells derived from healthy volunteers were isolated using Rosette Sep CD4+ enrichment cocktail (CAT#15022, StemCell® Technologies) (50 μL/ml of whole blood) according to the instructions of the supplier. To sort CD4+CD25-CD45RA- (Tmem) the purified CD4+ cells were washed and surface stained with anti-CD25-BV510® (M-A251, BD), anti-CD45RA- PE®(4KB5, Dako) and CD4-PE-Cy5.5® (13B8.2, Beckman-Coulter) and FACS-sorted on a FACSAria™ III machine (BD). FACS-sorted CD4+ Tmem cells (2.5 x 104 cells/well) were cultured in 96-well U-bottom plates and incubated with Dynabeads® Human T-Activator CD3/CD28 (1:5, bead:cell ratio) (Cat# 111.31D, Gibco®) and recombinant human IL-2 (rhIL-2) (100 U/mL) (Proleukin Prometheus Laboratories) for three days at 37C with 5% CO2. RPMI-1640 Dutch modified (Gibco®) culture medium, with sodium bicarbonate and 20 mM HEPES, supplemented with penicillin/streptomycin (100 U/mL), sodium pyruvate (1 mM), glutamine/glutamax and 10% human pooled serum (HPS, Radboudumc), was used in all conditions.] FACS-sorted CD4+ Tmem cultured as described in Protocol 2 (Growth protocol) were supplemented with recombinant human TNF-alpha (rhTNF-alpha, 50 ng/mL, CAT# 210-TA-020, R&D). On day three of culture the cells were pooled and RNA extraction was carried out. Total RNA was extracted from lysed CD4+ Tmem by using the RNeasy Plus Micro kit (Qiagen). Initial quality check and RNA quantification of the samples were performed by capillary electrophoresis using the LabChip GX (Perkin Elmer). Non-degraded RNA-samples were selected for subsequent sequencing analysis. Sequence libraries were generated using the TruSeq (polyA) mRNA sample preparation kits (Illumina) using the Sciclone NGS Liquid Handler (Perkin Elmer).
characteristics.characteristic: description
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: anti-CD3/CD28-coated bead (1:5, bead:cell ratio) stimulation + recombinant human IL-2 (100 U/mL)
characteristics.characteristic: growth condition
characteristics.text: Donor 3
characteristics.characteristic: individual
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: blood
characteristics.characteristic: organism part
characteristics.text: CD4+ CD25- CD45RA-
characteristics.characteristic: phenotype
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: rhTNF_alpha treatment_Donor 4
characteristics.characteristic: title