Validation of T cell development in Cas9-LPs at the single-cell level. (A) UMAP1-2 visualization of scRNA-seq data from Cas9-LP with or without Notch stimulation (Fig. 1 A, left). The color intensity represents the expression levels of Spi1, Tcf7, and Bcl11b, which indicate the progression of T cell development. (B) UMAP1-2 visualization of scRNA-seq data from Cas9-LP with or without Notch stimulation (Fig. 1 A, left). The color intensity indicates the expression levels of Rorc and Id3, which are informative for identifying Phase 3 cells. (C) UMAP visualization of scRNA-seq data for Cas9-LP with or without Notch stimulation (related to Fig. 1 A, left). The cells are colored according to the cell cycle stages. (D) UMAP visualization of scRNA-seq data for Cas9-LPs with and without Notch stimulation. Clustering was performed using a nonlinear representation of the top 50 principal components, excluding PC2, which contained many cell cycle–related genes. The cells are colored according to the time after Notch stimulation (left) or the pseudo-time scores (right). (E) Heatmap showing changes in the expression of TFs in Fig. 1 D across different developmental stages of DN cells. (F) Tag count distributions for RUNX1 ChIP signals are shown as peak-centered heatmaps. Each lane represents the merged tag directories from two biological replicates. (G) Heatmap showing changes in the motif enrichment of stage-specific RUNX1-binding genomic regions in Fig. 2, A and B. (H) Myc- and FLAG-tagged RUNX1-ERT2 were retrovirally transduced into Cas9-LPs, and cells were treated with tamoxifen for 6 h. Nuclear lysates from Mock- or Myc-FLAG-RUNX1-ERT2–expressing LPs, with or without tamoxifen treatment, were subjected to immunoblotting with anti-FLAG and anti-lamin B antibodies. Two independent experiments were performed with similar results. (I) Mascot scores of representative RUNX1-binding molecules in LP, Phase 1, and Phase 2 cells (Fig. 2 D) are shown. Source data are available for this figure: SourceData FS2.