(A) Validation of genotypes of E3.5 female embryos. Eed exon 7 RNA-Seq reads are normalized to total mapped RNA-Seq reads. (B) Table describing the RNA-Seq genotypes, number of sequenced embryos, average percentage maternal X-linked gene expression, average number of SNPs per X-linked gene, and the SNP overlapping read coverage threshold. A comprehensive list of expression levels of all informative genes is included in Supplementary file 2. (C) Normalized maternal or paternal reads per X-linked gene in individual Eedfl/fl, Eedfl/-, Eed-/-, Eedm-/-, and Eedmz-/- female E3.5 blastocysts. (D) Pyrosequencing-based quantification of allelic expression of X-linked genes Xist, Rnf12, Atrx, and Pgk1 in individual Eedfl/fl, Eedm-/-, and Eedmz-/- female E3.5 blastocysts. Error bars, standard deviation of data from 3 to 6 independent embryos. The mean allelic expression for Xist, Rnf12, and Atrx is significantly different between Eedfl/fl and Eedm-/- embryos (p<0.05, Welch’s two-sample T-test). The mean allelic expression for Xist, Rnf12, Atrx, and Pgk1 is significantly different between Eedfl/fl and Eedmz-/- embryos (p<0.05, Welch’s two-sample T-test). The mean allelic expression of Pgk1 is significantly different between Eedm-/- and Eedmz-/- embryos (p<0.05, Welch’s two-sample T-test). Pairwise statistical comparisons for all genes and between all genotypes are included in Supplementary file 4. (E) Allele-specific H3K27me3 ChIP-Seq at the Xist locus of wild-type MII oocyte, sperm, PN5 zygote, 8 cell embryo, and inner cell mass (ICM) (Zheng et al., 2016).