Single-cell RNA-seq of Nr4a3-Tocky post-positive selection thymocytes defines temporally anchored landmark populations during thymocyte development

This study generates Tocky landmark reference data to resolve the intersection of signaling history and developmental maturation of thymic T cells in single-cell state space. While single-cell RNA sequencing (scRNA-seq) provides high-dimensional snapshots of cellular heterogeneity, it often lacks an independent experimental record of biological time. We addressed this by integrating the Nr4a3-Tocky system, a fluorescent molecular clock of T-cell receptor (TCR) signaling, with droplet-based scRNA-seq of post-positive selection thymocytes. By flow-sorting and sequencing three discrete Timer-defined landmark populations (Blue+Red-, Blue+Red+, and Blue-Red+), we established an experimentally anchored temporal reference. This project maps the continuous transition of thymocytes toward CD4 and CD8 single-positive states within a shared canonical geometry, capturing the real-time dynamics of immediate TCR-response genes and lineage-associated regulators during agonist selection.