Extended Data Fig. 6: Loss of the E-repeat does not affect Xist abundance, splicing or stability.

a, RT–qPCR quantification of the fold upregulation of Xist^MS2 RNA during differentiation of wild-type or ΔE cells normalized against undifferentiated samples and an internal control (Rrm2). b, RT–qPCR measurements of Xist^MS2 RNA half-life (upon actinomycin D treatment) at day 3 of differentiation in wild-type or ΔE cells, calculated as MS2 transcript copy number per μg of total RNA. For a and b, error bars represent the s.e.m. (n = 3, measured in triplicate). Differences were not significant by two-tailed Student’s t-test. c, Epifluorescence images of differentiation day 4 wild-type and ΔE cells probed for exonic regions of Xist (red) or Xist intron 1 (yellow), and DAPI stained, indicating that the Xist^ΔE transcripts within the cloud are spliced. d, Same as Fig. 1h, except two additional Xist^ΔE-expressing nuclei are shown. Scale bar, 5 μm. e, Same as Fig. 1h, except for the nuclei in d. Note aberrant localization of Xist^ΔE at the nuclear lamina. f, 3D Amira reconstructions of the cells shown in Fig. 1h. g, Representative epifluorescence images of RNA FISH against Xist and MS2 with DAPI staining for comparison to super-resolution images in d, e and Fig. 1e, h. Inset, enhanced image of the marked area. h, Box plot showing the distribution of the area (in pixels) covered by the Xist RNA FISH signal, used to calculate the Xist aggregation score in Fig. 1f (n = 30, from one experiment). i, Same as h except showing distribution of the bounding circle area, (n = 30, from one experiment); ***P < 0.00005, two-sample Kolmogorov–Smirnov test. j, Box plot of the average distance between Xist foci within Xist-^MS2 clouds in differentiation day 7 wild-type and ΔE ES cells, as measured by IMARIS. 50 measurements were made per cell, 5 cells per sample; ****P < 0.000005, two-sample Kolmogorov–Smirnov test. For h–j, horizontal lines denote the median, whiskers indicate 1.5× the interquartile range, dots represent outliers.