RNA-sequencing.

Bulk RNA-seq was performed on 500 sort-purified OT-I T cells derived from OT-I memory mice after culture in conditions of 2 h no stimulation, 2 h stimulation with 100 ng/mL TGF-β, 24 h stimulation with 100 nM N4, and 24 h stimulation with 100 nM N4 and 100 ng/mL TGF-β. Twenty-four-hour stimulation stain control was performed to ensure T cell activation occurred consistently with prior experiments (SI Appendix, Fig. S7A). In total, 28 samples were sequenced, and each condition was represented by a total of seven biological replicates (combined from two independent experiments). Cells were prepared for RNA sequencing, and data were overall analyzed as previously described (57) aside from using the GRCm38 reference genome.