iTEC/thymocyte organoids were homogenized with pestles and cells were washed in buffer consisting of 1% FBS in PBS. Cells were stained with antibodies diluted 1:10 in the same buffer on ice for 20 min, and, after one more wash, they were analyzed with the BD FACSAria II. The CD45+CD3+TCRab+CD4+ and CD45+CD3+TCRab+CD8a+CD8b+ populations were sorted and resuspended in ISOGEN-LS. RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen). For donor-derived controls, thymocytes were isolated from homogenized 2-3 mm thymus pieces, whose supernatant cells were incubated in RBC Lysis Buffer (BioLegend) for 10 min on ice and frozen before being subjected to the same staining, sorting, and RNA extraction protocols as the iTEC-supported thymocytes. RNA was reverse transcribed with Superscript III reverse transcriptase (Thermo Fisher), after which TCRb genes were amplified with adaptor-ligation PCR. Index sequences were added using the Nextera XT Index Kit v2 Set A or Set D (Illumina).