RNA-seq results from mouse T cell precursors in different developmental stages including DN1, DN2a, DN2b, DN3, and DP [Gene Expression Omnibus accession: GSE89198; (58)] were used to create DN1 and DN2 gene sets. Of this RNA-seq dataset, log2-transformed FPKM (Fragments Per Kilobase Million) values of 25 DN2 and 8 DN3 WT mice were used for differential expression analysis with Limma. Genes that were differentially up-regulated [P < 0.05 and log fold change (FC) > 2] between DN1 versus DN3 (365 genes), DN2a versus DN3 (342 genes), DN2b versus DN3 (120 genes), and DN2a/b combined versus DN3 (141 genes) were used as gene sets for GSEA. GSEAPreranked (GSEA 4.0.3, Broad) was run on all expressed WT versus TCF−/− RNA-seq genes, which were ranked by the P value and log FC generated by DESeq2. DN3b TCF−/− was negatively associated with the DN1 gene set [normalized enrichment score (NES) of −1.04] and positively associated with all of the DN2 gene sets (DN2a NES 1.23, DN2b NES 1.53, and DN2a/b combined NES 1.36).

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