title: Illumina NovaSeq 6000 sequencing; Transcriptional profiling of human in vitro primed Th1, Th2, Th9 and Treg T cells
design: Transcriptional profiling of human in vitro primed Th1, Th2, Th9 and Treg T cells
library_construction_protocol: Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. Human naïve CD4+ T Cell Isolation Kit Human (Stemcell Technologies) as per the manufacturer's instructions. Naïve T cells were stimulated with anti-CD3/CD2/CD28 beads (T cell/bead = 2:1, Miltenyi) and primed into effector CD4+ T cell subsets with IL-12 (5 ng/ml) (BioLegend) for TH1 cells, IL-4 (50 ng/ml) (BioLegend) for TH2 cells, IL-4 (50 ng/ml) and TGF-b (5 ng/ml) (R&D Systems) for TH9 cells, and TGF-b (5 ng/ml) for iTREG. From cell culture initiation to analysis, the culture medium was supplemented with the indicated cytokines every other day.  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 Naïve T cells were stimulated with anti-CD3/CD2/CD28 beads (T cell/bead = 2:1, Miltenyi) and primed into effector CD4+ T cell subsets with IL-12 (5 ng/ml) (BioLegend) for TH1 cells, IL-4 (50 ng/ml) (BioLegend) for TH2 cells, IL-4 (50 ng/ml) and TGF-b (5 ng/ml) (R&D Systems) for TH9 cells, and TGF-b (5 ng/ml) for iTREG. Total RNA was isolated with RNAeasy kit (Qiagen) Illumina TruSeq Stranded mRNA protocol (Directional, first strand)