GSM6185953: RNA-Seq ST2+ Th2 Replicate 3; 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 or 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, live ST2+ CTLs, Th1 and Th2 cells were flowcytometrically sorted and subjected to RNA isolation. 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. TrueSeq stranded total RNA library kit (Illumina)