alias: E-MTAB-5622:rhTNF_alpha treatment_Donor 5_s
title: Illumina HiSeq 2500 sequencing; Transcriptional profiling of activated CD4+ memory T cells exposed to anti-TNF and recombinant human TNF-alpha in vitro
design: Transcriptional profiling of activated CD4+ memory T cells exposed to anti-TNF and recombinant human TNF-alpha in vitro
library_construction_protocol: 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).
identifiers.namespace: Radboud University Medical Center (Radboudumc)
identifiers.id: E-MTAB-5622:rhTNF_alpha treatment_Donor 5_s