Wild-Type vs Orai1KO pTH17 and npTh17 cells_ 48h
Transicriptome comparison between WT and ORAI1 KO pTH17cells at 48 hours during differentiation Overall design: Naive CD4+ T-cells were isolated and purified from either wild-type or ORAI1 KO mice. Naïve CD4+ T cells were enriched by magnetic sorting from single-cell suspensions generated by mechanical disruption of spleens and lymph nodes of adult mice using MagniSort naïve CD4+ T cell enrichment kit (catalog # 8804-6824-74,ThermoFisher Scientific). For effector T cell differentiation, cells were stimulated with anti-CD3 antibody (1452C11, Bio X Cell) and anti-CD28 antibody (Clone 37.51, Bio X Cell) for 48 hours on a plate coated with rabbit anti-hamster antibody (MP Biomedicals). CD4 + CD25 - T cells were cultured with anti-IL-4 antibody (Peprotech) and 10 µg/ml anti-IFN-? antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1ß (R&D Systems) for pathogenic Th17 differentiation and cultured in T cell medium. For non-pathogenic Th17 cell differentiation,CD4 + CD25 - T cells were cultured with 10 µg/ml anti-IL-4 antibody, 10 µg/ml anti-IFN-? antibody, 30 ng/ml IL-6 (Peprotech) and 1 ng/ml TGF-ß (Peprotech). Cells were taken off the plate on D2 (48hrs) and prepared for bulk RNA sequencing analysis.