Thymi were extracted from C57Bl/6J mice (11-13 weeks old, 2 males, 2 females), and dissociated with collagenase D to maximally release lymphoid and non-lymphoid cell-types. Each thymus was stained (CD45, CD3e, CD4, CD8, DNA-tagged barcodes – each mouse tagged individually) per Immgen SOP (https://www.immgen.org/img/Protocols/ImmGenCellPrepSortingSOP.pdf). Thymocytes were sorted to rebalance subpopulations in the final sample (20% total CD45+ thymocytes, 47% CD45+CD3hi, and 33% CD45+CD4-CD8- double negatives). The sorted cells were immediately taken for encapsulation and single-cell RNAseq using the 10x 3’v3.1 protocol.

Additional info at https://www.immgen.org/. For the protocol used to prep these samples, check the Cell Prep and Sorting SOP (link). For a guide on referencing the data, see the Data requests section (link).