Extended Data Fig. 1: SHARP enrichment over the Xi increases in a non-stoichiometric manner relative to Xist.

a) Schematic of dox-inducible Xist expression system. The endogenous Xist promoter is replaced with a TetO element that can be activated upon the addition of doxycycline. b) Percent of cells expressing zero, one, or two alleles of the silenced Atrx gene as measured by RNA-FISH at various timepoints after Xist induction. c) Illustration of SHARP enrichment analysis over the Xi in TX-SHARP-HALO female mESCs. The Xi region was demarcated based on Xist RNA-FISH; SHARP was demarcated by either direct HALO labelling or immunofluorescence (anti-HALO). Fluorescent intensities of RNA-FISH probes, HALO tag, or anti-HALO immunofluorescence were then quantified within the defined Xi region and plotted. d) Representative images of Xist and SHARP localization in TX-SHARP-HALO female mESCs across 72 hours of Xist expression (n > 15 cells per timepoint from two independent experiments); Xist visualized by RNA-FISH (magenta); SHARP visualized by immunofluorescence labelling with anti-HALO antibody (green). Images shown as max. projections; scale bars 10 μm. e) Quantification of total fluorescence intensity of Xist (RNA-FISH) in multiple individual cells over 48 hours of Xist expression (Fig. 1b). f) Quantification of Xist and SHARP intensities in multiple individual cells over 72 hours of Xist expression (Extended Data Fig. 1d). Top panel: area of the territory coated by Xist RNA (µm2); middle panel: average fluorescent intensity of Xist (RNA-FISH) per unit within the Xist territory; bottom panel: average fluorescent intensity of SHARP (anti-Halo antibody) per unit within the Xist territory. g) Representative images of Xist and SHARP localization in TX-SHARP-HALO female mESCs after 48 and 72 hours of RA-induced differentiation (n > 10 cells for each timepoint); Xist visualized by RNA-FISH (magenta), SHARP visualized by direct Halo labelling (green). Images shown as max. projections; scale bars 10 μm. h) Quantification of Xist and SHARP in individual differentiated cells (Extended Data Fig. 1g). Left panel: area of the territory coated by Xist RNA (µm2); top right panel: average fluorescent intensity of Xist (RNA-FISH) per unit within the Xist territory; bottom right panel: average fluorescent intensity of SHARP (direct Halo labelling) per unit within the Xist territory.