Extended Data Fig. 7. Representative fluorescence-activated cell sorting (FACS) gating strategy for E11.5 WT and cRE1^dup/+ Isl1^MNGFP⁺ r3-r7 cranial motor neurons.

Gating strategy for dissociated GFP-free limb buds collected from E11.5 WT;Isl1^MN-GFP embryos (a-e) and r3-r7 hindbrains collected from E11.5 WT (f-j) and cRE1^dup/+ (k-o) embryos. (a,f,k) P1 was drawn to include all cells and exclude debris and dead cells based on SSC-A (side scatter area) VS FSC-A (forward scatter area). (b,g,l) P2 was drawn for primary doublet removal using the ratio of FSC-H (forward scatter height) vs FSC-A to exclude doublets entering the point of interrogation vertically. (c,h,m) P3 was drawn as a secondary exclusion for horizontal doublets using the side scatter parameter of SSC-H (side scatter height) vs SSC-W (side scatter width). (d,i,n) GFP positive gate was drawn to include true GFP positive cells and exclude any possible autofluorescent signals from live or dead cells. GFP signal was plotted against autofluorescence (autoFl) detected as a second channel from the GFP laser and an emission filter of 575/40. (e,j,o) Gating summary, GFP⁺ cells comprised 0% of WT limb bud input, 2.0% of WT input hindbrain cells, and 2.3% of cRE1^dup/+ input hindbrain cells.