Polyclonal human TCRvβ development in thymocytes differentiated from shRNA 1005-transduced HPSCs. Total RNA isolated from FACS-purified EGFP⁺ or mCherry⁺ thymocytes taken from thy/liv organoids of 3 reconstituted BLT mice were subjected to a quantitative PCR-based TCR spectratyping analysis. Peak ratio was compared with the median of each TCR Vβ family between EGFP⁺ and mCherry⁺ thymocytes. There was no significant difference between the 2 groups (P = .668, by Mann-Whitney test). TCRs are made from splicing of V-D-J regions, and the complementarity determining region-3 region is therefore random in size. Thus, the PCR output bands corresponding to different sized splice products, and the intensity of these bands should be Gaussian in distribution if T-cell repertoires are random.