Illumina HiSeq 2500 paired end sequencing; Time series RNA-seq of Naïve CD4 positive T-cells undergoing differentiation towards T-helper 1.
Time series RNA-seq of Naïve CD4 positive T-cells undergoing differentiation towards T-helper 1.
Naïve T-cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic bead separation using a Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec). The PBMCs were isolated from the buffy coat fraction of human blood using phase separation over lymphoprep (Axis Shields). Stimulation towards Th1 differentiation for 0.5, 1, 2, 6 and 24h. Specifically the cells were treated with a 1/1 ratio of Dynabeads® Human T-Activator CD3/CD28 (Thermofisher), recombinant IL-2 (10 ng/ml), recombinant IL-12 (5 ng/ml) and anti-IFNG (MAB285) (5 µg/ml) (Rnd Systems) in RPMI 1640 media (Gibco). RNA (and DNA) was isolated with a ZR-duet DNA/RNA miniprep kit (Zymo Research) according to the manufactures instructions. Samples were processed using the TruSeq RNA Sample Prep v3 kit (Illumina) according to the manufacturers instructions.