Targets of EOMES in mouse CD4+ T cells

To gain mechanistic insights into how EOMES regulates CD4+ T-cell differentiation and function, we performed an RNA-seq analysis using Eomes-GFP reporter mice (Eomes+/GFP) to isolate GFP+ (EOMES+) and GFP- (EOMES-) CD4+ T-cells by FACS sorting. We also sequenced RNA from Eomes-deficient GFP+ and GFP- CD4+ T-cells isolated from EomesΔT/GFP knock-out mice, in which one Eomes allele is disrupted by GFP insertion and the DNA-binding domain of the other allele is deleted in T cells by Lck-driven Cre recombinase. Hence, we had two populations of GFP+ cells where the EOMES locus was transcribed, one with a transcriptionally active EOMES protein (Eomes+/GFP) and another one with no transcriptionally active EOMES (EomesΔT/GFP), plus the respective GFP-negative controls isolated from the same mice. A homogeneous population of EOMES-expressing CD4+ T cells were obtained by transferring naïve sorted CD25- CD45RBhigh CD4+ T-cells isolated from Eomes+/GFP reporter or EomesΔT/GFP knock-out donor animals into Rag2-/- mice. After three weeks of adoptive transfer GFP+ and GFP- CD4+ T-cell populations from these mice were FACS-sorted for transcriptome analysis by RNA sequencing. Altogether we submit 12 samples, representing 4 biological conditions (Het_GFP_neg, Het_GFP_pos, KO_GFP_neg and KO_GFP_pos), 3 replicates each. The most relevant comparisons are (1): Het_GFP_pos vs Het_GFP_neg, which reveals the transcriptional signature of Eomes-expressing CD4 T cells, and (2): Het_GFP_pos vs KO_GFP_pos, which reveals Eomes-dependent genes. GFP positive and negative cells isolated from the same recipient have the same replicate number (e.g., Het replicate 1).