IR-seq analysis of T cell developmental stages confirms that Jα-Jα ECs originate during alpha chain rearrangement. (A) Schematic diagram of T cell development showing stage-specific, thymus-derived cell sorts used to generate 12 IR-seq libraries (see Methods). (B) Coverage in the intermediate CD4 single positive (ISP) IR-seq library across the Tcra locus. Coverage was computed between neighboring Vα, Dα, and Jα gene segment RSSs and normalized relative to coverage in the same regions in the prerecombinational DN1 IR-seq data set. Depleted coverage across the Tcr delta locus is predicted to result from genomic excision events and loss of delta ECs during preceding proliferative phases. Coverage across the Jα subregion remains at ∼1, indicating that Jα-Jα ECs are not overrepresented in IR-seq. (C) Heatmap analyses of sorted T cell precursors showing Vα-Jα EC events (top) and Jα-Jα EC events (bottom) for eight consecutive recombinationally active IR-seq libraries. (D) Jα-Jα–to–Vα-Jα (blue bars) and Vα-Vα–to–Vα-Jα (red bars) ratios of RFs in 12 IR-seq libraries, representing key stages of T cell development. (E) DP-to-RF ratios for Vα-Jα (blue bars) and Jα-Jα (red bars) in 12 IR-seq libraries representing key stages of T cell development.
