Illumina HiSeq 4000 paired end sequencing; Time series RNA-seq of Th1 differentiation: The effect of Progesterone on Th1 differentiation?
Time series RNA-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), anti-IFNG (MAB285) (5 µg/ml) (Rnd Systems) and 50 µM Progesterone (P4) in RPMI 1640 media (Gibco). Prior to start of stimulation cells were preincubated for 12-14h with 50 µM P4. RNA was extracted using the ZR-Duet DNA/RNA Kit (Zymo Research, #D7001) and nucleic acid concentration was determined by nanodrop. RNA used for RNA sequencing underwent treatment with 5 U DNase I (Zymo Research, #E1010) in column during RNA extraction. Only RNA samples with a RNA integrity number > 7 were chosen for RNA sequencing as shown by 2100 Bioanalyzer (Agilent). Samples were processed using the TruSeq RNA Sample Prep kit (Illumina) according to the manufacturers instructions.