title: Illumina HiSeq 3000 sequencing; Transcriptional profiling of Th1, Th2, Th9, and Th17 T cell clones
design: Transcriptional profiling of Th1, Th2, Th9, and Th17 T cell clones
library_construction_protocol: Human CD4+ T cells were isolated from PBMC by EasySep positive selection kit (Stemcell Technologies) according to the manufacturer's instructions. Positively selected CD4+ T cells were stained for subsequent TH cell subset sorting. Total RNA was isolated from TH1, TH2, TH17, and TH9 clones in the resting state with RNAready-kit (AmpTec) according to manufacturer's instruction. Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation.  Culture medium consisted of PRMI-1640 with Hepes (GIBCO) supplemented with 5% of heat-inactivated human serum (Swiss Red Cross, Basel, Switzerland), 2mM L-Glutamine (Biochrom), 50U/ml penicillin and 50μg/ml streptomycin (Bioconcept) and 50 IU/ml IL-2 (Roche). T cells were cultured at a density of 0.25-1x105cells/96 well plate in a total volume of 200μL cell culture medium.  Illumina TruSeq Stranded mRNA protocol Illumina TruSeq Stranded mRNA protocol
attributes.tag: Experimental Factor: cell type
attributes.value: T-helper 2 cell
attributes.tag: Experimental Factor: time
attributes.value: 9
attributes.tag: Experimental Factor: stimulus
attributes.value: antibodies against CD3, CD2, and CD28