Fig 2: Study strategy and ILC-related populations analysis overview.a Schematic overview of the strategy of this study. Cells from human fetal hematopoietic (liver), lymphoid (thymus and spleen) and non-lymphoid (intestine, skin and lung) tissues at indicated gestational stages were first labeled with cell hashing antibodies and fluorescence-labeled antibody for sorting based on the sorting strategy in e. The cells from 2–6 tissues were pooled and then loaded on a droplet-based 10× genomics scRNA-seq platform. Cells of each tissue from different samples were distinguished by cell hashing barcode. The information of individual sample was listed in Supplementary information, Fig. S1. Two replicates per developmental stage, with a total of 6 samples were used in this study. b The gating strategy to identify human mature ILCs with example from 12 PCW thymus. In the 7AAD– lineage (CD3, CD4, CD5, FcεRI, CD11c, CD11b, CD14 and CD19)-negative (Lin–)CD34-CD45+ cells, there are CD56+CD127– killer ILCs and CD56–CD127+ helper ILC subsets. The helper ILCs are divided into CRTH2+ ILC2, CRTH2–CD117+ ILC3 and CRTH2–Kit– putative ILC1. c Pie charts show the proportions of three mature helper ILCs of each organ based on flow cytometry analysis. The proportions were calculated based on more than three independent experiments. d Sorting strategy of human fetal lymphoid progenitors and ILC-associated populations. In the 7-AAD–Lin–CD45+ cells, CD34+CD127+ lymphoid progenitors, CD34+CD127– HSPCs, CD34–CD161+CD127+/– ILCs and CD34–CD161– cells were sorted from each tissue, and mixed at the ratios indicated in Supplementary information, Fig. S1 for scRNA-seq. e The flow cytometry results show that the percentages of the cell populations identified in d in each tissue change from 8 to 12 PCW. The results are representative of 2–3 independent experiments in each gestational week.