RNA-seq analysis of WT and Tmem11 KO Th1 cells

Loss of TMEM11 did not influence T cell differentiation but selectively impaired effector functions of Th1 cells, resulting in reduced severity of symptoms in an animal model of autoimmunity. To find out genes influenced by Tmem11 knockout, we conducted RNA-seq analysis using Th1 cells after re-stimulation. Overall design: Naive CD4+ T-cells were isolated and purified from either wild-type or TMEM11 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 IL-12 for Th1 differentiation. After detaching from the plate, cells were further cultured for 48 hours and re-stimulated with anti-CD3 and anti-CD28 antibodies. Bulk RNA sequencing analysis was performed to identify transcriptomic changes associated with loss of TMEM11 in Th1 cells.