Extended Data Fig. 5: ΔE ES cells undergo differentiation similar to wild-type ES cells and splicing of Xist-intron 6 proceeds in the absence of the E-repeat.
From: A protein assembly mediates Xist localization and gene silencing
a, Homologous recombination strategy used to delete the Xist E-repeat in female ES cells. b, Southern blot strategy with a 5′ external probe for identification of deletion clones. c, Southern blot (described in b) on targeted ES cells with a loxP-flanked puromycin cassette in place of the E-repeat on one Xist allele. d, Sequencing analysis (black) of the wild-type Xist-PCR amplicon in ΔE cells (red line in b). 129-allele SNPs are shown in red and do not match those in PCR amplicon, confirming E-repeat deletion on the XistMS2(129) allele. e, Tsix RNA FISH on undifferentiated wild-type and ΔE ES cells confirms the presence of two Tsix nascent transcription units, used as a proxy to confirm targeted cells maintain two X chromosomes. f, Bright-field images of wild-type and ΔE cells at day 4 of differentiation, showing that differentiating cells are morphologically similar. g, Immunoblot of differentiation day 2 wild-type and ΔE cell lysate, showing equal loss of NANOG expression. h, Sequence of genomic and cDNA amplicons of the XistΔE allele after puromycin cassette removal, confirming correct targeting and the use of a cryptic splice site in ΔE cells. i, Exon 6–7 RT–PCR amplicons generated from RNA isolated from day 4 differentiated wild-type (primers APJ248/624) or ΔE (primers APJ248/631) cells. The ΔE PCR amplicon was shorter than expected. Sequencing revealed a cryptic 3′ splice site downstream of the loxP site that extended the E-repeat deletion within the Xist transcript (but not the Xist genomic DNA) by 42 nt (see (h)). j, PCR amplicons from wild-type or ΔE genomic DNA using the same primers as in i. The intron 6-containing products can be amplified, indicating non-detection of intron 6-containing Xist transcripts is not due to amplification problems. k, Schematic outlining primers used to assess Xist DNA and RNA in i and j. For c, g, i and j, see Supplementary Fig. 1 for source data.
