Illumina NovaSeq 6000 paired end sequencing; Time series ATAC-seq of Th1 differentiation: The effect of Progesterone on Th1 differentiation?
Time series ATAC-seq of Th1 differentiation: The effect of Progesterone on Th1 differentiation?
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). For the Progesterone treated samples: Sampels were pre-incubated with 50 µM Progesterone (P4) for 12-14h prior to experiment and then reconstituted with fresh media containing above 50 µM P4 and above mentioned TH1 stimulation reagetnts to start the reaction. Non-progeterone treated sample were pre-incubated and reconstituted in growth media without P4. DNA was extracted from 50000 cells per sample in accordance with the OMNI-ATAC-seq method. Corces et al. Nature Methods 2017 Libarary construction was carried out in accordance with the OMNI-ATAC-seq method. Corces et al. Nature Methods 2017. Illumina NEXTERA primers and sequencing adapters were used to construct the index.