SRP492124 PRJNA1081449 GSE259418 Thymic mimetic cells in zebrafish We profiled the zebrafish thymus by single-cell RNA sequencing to investigate the heterogeneity of zebrafish thymic epithelial cells and determine whether similar mimetic cell populations to those found in mice and humans might also exist in the zebrafish. Overall design: To assess the heterogeneity of zebrafish thymic epithelial cells, a total of 11 zebrafish samples derived from 14 adult and 21 juvenile zebrafish were studied. Specifically, we re-processed single-cell RNA sequencing (scRNA-seq) data from 8 zebrafish thymus samples derived from 4 adult zebrafish and 21 juvenile zebrafish previously described in GSE190794 (GSM5732067, GSM5732070, GSM5732077, GSM5732078, GSM5732079, GSM5732080, GSM5732081, GSM5732082) so that reads mapping to intronic regions were included in the analysis to increase sensitivity and be consistent with mouse and human scRNA-seq studies for cross-species comparison. In addition, we generated 3 new scRNA-seq datasets derived from a total of 10 adult lck:mCherry;cd79a:eGFP transgenic zebrafish. The thymi from 5 of these adult zebrafish were subjected to mechanical dissociation, filtering, and cell sorting on a BD FACSAriaII, whereas thymi from the other 5 adult zebrafish underwent enzymatic dissociation with Liberase TM prior to quenching, filtering, and cell sorting. The live, non-autofluorescent, GFP-negative, mCherry-negative cells (a population depleted of T and B cells, thus providing a relative enrichment of thymic epithelial cells) were sorted and collected for 10x scRNA-seq. One 10x lane was used for the mechanically dissociated sample (untreated) and two lanes were used for the liberase-treated sample to obtain 2 technical replicates. Re-analysed Samples in GSE190794/PRJNA788489 and their processed data file: SampleName Experiment BioSample processed data file GSM5732067 Adult, Thymus, 3 SRX13397695 SAMN24007722 Fish_22_Thymus_.filtered_feature_bc_matrix.h5 GSM5732070 Adult, Thymus, 4 SRX13397698 SAMN24007725 Fish_24_Thymus_.filtered_feature_bc_matrix.h5 GSM5732077 Adult, Thymus, 1 SRX13397705 SAMN24007732 lck-eGFP_Thymus_1_.filtered_feature_bc_matrix.h5 GSM5732078 Adult, Thymus, 2 SRX13397706 SAMN24007733 lck-eGFP_Thymus_2_.filtered_feature_bc_matrix.h5 GSM5732079 Juvenile, Thymus, 1 SRX13397707 SAMN24007734 4wpfThymus_1.filtered_feature_bc_matrix.h5 GSM5732080 Juvenile, Thymus, 2 SRX13397708 SAMN24007735 4wpfThymus_3.filtered_feature_bc_matrix.h5 GSM5732081 Juvenile, Thymus, 3 SRX13397709 SAMN24007736 4wpfThymus_5.filtered_feature_bc_matrix.h5 GSM5732082 Juvenile, Thymus, 4 SRX13397710 SAMN24007737 4wpfThymus_6.filtered_feature_bc_matrix.h5 GSE259418