alias: GSM2464963
title: GSM2464963: Sample_deg_1hrs; Mus musculus; RNA-Seq
library_construction_protocol: Murine CD4 positive cells were isolated from spleen cells by magnetic bead separation using EasySep mouse CD4+ cell negative selection kit (StemCell Technologies) according to the manufacturer’s instructions. The purity of isolated CD4+ cells was routinely exceeded 95%. For activation, CD4+cells were cultured at 5x106 cells/ml coated with 5µg/ml anti-CD3, and added 1µg/ml soluble anti-CD28 in complete RPMI and analysed at 4 and 20h. Th0, Th1 and Th2 skewing cultures, CD4+ cells were cultured on 5ug/ml plate bound anti-CD3 with were culture with 1µg/ml soluble anti-CD28 in complete RPMI. Th1 conditions the solution containing 5µg/ml anti-IL-4 and 10ng/ml rmIL-12. For Th2 cultures, 5µg/ml anti-IFNɣ; 20ng/ml rmIL-4, and 5µg/ml anti-IL-12 were added and, Cells were cultured at 37 °C under 5% CO2. Cells were harvested 72h and T-bet and Gata-3 were analysed through intracellular staining assay as described (Furmanski et al. 2013) (antibodies/proteins: eBioscience). RNA libraries were prepared for sequencing using standard Illumina protocols
identifiers.namespace: GEO
identifiers.id: GSM2464963