library_construction_protocol: Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without calcium or magnesium at room temperature. Live cells were sorted (85 um nozzle) on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3' v3.1 Reagent Kits (10x Genomics, Inc.). A targeted recovery of between 4,000 and 6,000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 User Guide (Rev D). The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core (https://bauercore.fas.harvard.edu/) on an Illumina NovaSeq 6000 (Read 1: 28 cycles, i7 index: 8 cycles, i5 index: 0 cycles, Read 2: 91 cycles).