Extended Data Fig. 3: CITE-seq and fluorescence-based flow cytometry reveal the timing of expression for transcription factors and other features of CD4-CD8 T cell development.

a, Expression over pseudotime of Cd69 (dashed) and CD69 (solid). Features are totalVI denoised expression values scaled per feature and smoothed by loess curves. b, Expression of RNA and protein features over pseudotime by genotype. Features are totalVI denoised expression values scaled per feature and smoothed by loess curves. A resource of protein and RNA expression over pseudotime for all differentially expressed features is in Supplementary Information. c, Transcription factor protein expression in adult thymocyte populations. Representative histograms displaying GATA3, THPOK, and RUNX3 transcription factor expression detected by intracellular flow cytometry staining in CD4-fated (MHCI^-/-) and CD8-fated (MHCII^-/-) thymocyte populations. Thymocyte populations were gated on lymphocytes, excluding forward scatter and side scatter doublets, live cells, TCRβ⁺CD5^int/hi then on CD4 versus CD8. Cell populations were gated into the following subsets based upon cell surface marker expression: DP1 (CD4⁺CD8⁺CD127⁻CD69⁻), DP2 (CD4⁺CD8⁺CD127⁻CD69⁺), DP3 (CD4⁺CD8⁺TCRβ^hiCD5⁺CD127⁺CD69⁺), CD4⁺CD8^lo (CD4⁺CD8^loCD69⁺), semimature CD4 (CD4 SM, CD4⁺CD8⁻CD69⁺), mature CD4 (CD4 Mat, CD4⁺CD8⁻CD69⁻), semimature CD8 (CD8 SM, CD8⁺CD69⁺), and mature CD8 (CD8 Mat, CD8⁺CD69⁻). Data is concatenated from n = 4 mice per genotype. Positive staining was determined using a fluorescence minus one control. d, Expression over pseudotime of the Hallmark Il2-Stat5 Signaling signature⁷⁴ displayed as the mean of scaled totalVI denoised expression per gene, smoothed by loess curves.