Wild-Type vs ORAI 2/3 DKO vs ORAI 1/2/3 TKO_Pathogenic Th17 cells_ 48h

Transicriptome comparison between murine WT, ORAI 2/3 DKO, and ORAI TKO pTH17cells at 48 hours following differentiation Naive CD4+ T-cells were isolated and purified from wild-type (control), ORAI 2/3 DKO, and ORAI 1/2/3 TKO 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-γ antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1β (R&D Systems) for pathogenic Th17 differentiation in T cell medium. Cells were collected at 48h and prepared for bulk RNA sequencing analysis.