GSM6185967: RNA-Seq Th1 2h +IL-33 Replicate 2; Mus musculus; RNA-Seq
Naive CD44- CD62L+ CD8+ P14 cells or CD44- CD62L+ CD4+ Smarta cells were flowcytometrically sorted from LCMV-TCRtg mice and differentiated in the presence of irradiated splenocytes from TCRbd-/- mice and cognate LCMV GP33-41 (P14) or GP64-80 peptide (Smarta) peptide (1µg/ml). T cells were cultivated in RPMI1640 + GlutaMax I (Thermo Scientific) supplemented with fetal calf serum (FCS) (10%), penicillin (100 U/ml), streptomycin (100 μg/ml), gentamycin (10 μg/ml) and b-mercaptoethanol (50 ng/ml). For CTL and Th1 differentiation, IL-12 (5 ng/ml), IL-2 (5 ng/ml), and anti-IL-4 antibody (11B11, 10 μg/ml) were added. For Th2 differentiation, IL-4 (5 ng/ml), IL-2 (5 ng/ml), anti-IL-12 (C18.2, 10 μg/ml) and anti-IFN-g antibody (XMG1.2, 10 μg/ml) were added. T cells were harvested at day 5 of culture using Histopaque density centrifugation and cultivated for additional 5 days in identical culture conditions.  At day 10 of culture, T cells were harvested and rested for 3 days in the presence of IL-2 (5 ng/ml) and IL-7 (5 ng/ml), without irradiated splenocytes or cognate peptide. At day 13, IL-12 (5 ng/ml) was added to CTLs and Th1 cells to induce ST2 expression. At day 14 of culture, dead cells were removed using Histopaque density centrifugation. Subsequently, 2,5x105 T cells were plated in 96 flat bottom well plates and stimulated with IL-33 (10ng/ul) or left untreated. See Sorting strategy RNA was purified using the Nucleospin RNA XS Micro kit (Macherey & Nagel) according to manufacturer's instructions, without addition of carrier RNA. Smart-Seq v4 mRNA Ultra Low Input RNA Kit (Clontech)