knockdown on gene expression. (A) Similar distributions of expression fold changes for 342 X-linked (X) and 11,182 autosomal genes (A) with ≥1 RPKM in Patski cells between control and stable Firre knockdown. (B) Scatter plots of SNP-read counts from 434 alleles on the Xi (left panel) or on the Xa (right panel) between control and Firre stable knockdown in Patski cells. Only X-linked alleles with at least one SNP-read count in control or knockdown are included. Trend lines and R2 are shown. (C, D) Percent of genes upregulated (C) or downregulated (D) after Firre knockdown in Patski cells. X-linked genes are grouped as ‘subset 1’ representing those with ≥5 SRPM (SNP reads per 10 M mapped reads) from the Xi, and ‘subset 2’ representing those with <5 SRPM. (E) Expression changes for Firre, Xist, four escape genes (Kdm5c, Kdm6a, Shroom4, and Eif2s3x), and five genes subject to XCI (Atp7a, Pgk1, Iqsec2, Magee1, and Suv39h1) after Ctcf or Firre KD (siRNA or si&shRNA treatment). Expression ratios between KD and control measured by qRT-PCR (normalized to β-actin) or RNA-seq. Error bars, s.e.m. Genes showing significant expression changes by RNA-seq between Firre KD and control cells are indicated (***P <0.001 from Cuffdiff2 analysis). (F) No reactivation of Atp7a, Pgk1, and Iqsec2 was observed after Ctcf or Firre KD in Patski cells. Gel images of RT-PCR products without (-) and with (+) restriction enzyme (RE) digestion to specifically cleave BL6 alleles in BL6 cells, untreated Patski cells, and Patski cells treated with control siRNA, Ctcf siRNA or Firre siRNA are shown. (G) Expression changes for Xist, Nanog, and G6pdx in female mouse ES cells PGK12.1 during 14-day EB (embryoid body) differentiation. Expression measured by qRT-PCR normalized by 18S, and set to 1 in control cells at D0.