Two different sorting schemes were used. In a first experiment with n = 2 mice (sorting scheme 1), we purified 384 Lineage− (Lin−) KithiSca-1hi (LSK) cells with high surface expression of Kit and Sca-1 (encoded by Ly6a), a permissive sorting strategy to sample the pool of HSCs and multipotent progenitors. In addition, we sorted 768 lymphoid-primed multipotent progenitors (LMPP) as Flt3+ LSK cells, and 384 Lin−KitloSca-1lo as well as 192 Lin−KitloSca-1lo/−Flt3hi common lymphoid progenitors (CLP) in order to enrich for the lymphoid branch. We deliberately did not gate on Il7r to comprehensively sample CLPs1–3. In order to capture more mature and potentially underrepresented progenitor states of all hematopoietic lineages we subsequently applied a second sorting strategy (sorting scheme 2). Here, we sequenced 1,152 cells from 16 adjacent windows spanning different ranges of Kit and Sca-1 expression and being either Flt3+ or Flt3−. Cells in (a) and (b) were sorted from n = 2 mice each in another independent experiment. Flow cytometry data are from one experiment with n = 1 mouse.
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