Targeted nanopore DNA sequencing of mouse naive CD4 T cells activated under Treg polarizing conditions

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways and intracellular metabolites. Here we show that cell-permeable a-ketoglutarate (aKG) alters the DNA methylation profile of naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating FoxP3+ Treg differentiation and increasing inflammatory cytokines. Overall design: 5-methylcytosine (5mC) was studied in two independent experiments with 3 conditions each: thymic Tregs (tTregs), and in vitro CD4 T cells differentiated under Treg polarizing conditions (Tregs) with or without treatment with dimethyl ketoglutarate (DMK). Samples within each experiment were barcoded and multiplexed before Cas9 enrichment and nanopore sequencing.