Extended Data Figure 6. Characterization of the X^GX^T ESC line.

(a) Fluorescence microscopic images of X^GX^T ESCs. GFP/tdTomato double-positive cells (yellow cells in Merge 1 and 2) indicate two active X chromosomes while GFP⁺Tomato⁻ colony depicts cells that lost one of the X chromosomes.

(b) GFP/tdTomato double-positive X^GX^T ESCs were sorted at passage 5 and plated in S/L (p6) before measuring the percentage of double-positive and single-positive cells at p8, 10, 13 and 16 using flow cytometry.

(c) Karyotype analysis of undifferentiated, X^GO (GFP-positive) or X^TO (tdTomato-positive) ESCs (left) and GFP/tdTomato double-positive X^GX^T ESCs (right). GFP/tdTomato double-positive X^GX^T ESCs were sorted at passage 5 and maintained in S/L for 9 passages before analyzed. This result confirms that the progressive loss of GFP or TdTomato signal was due to the loss of an X and not X-inactivation due to differentiation.