Escape from X inactivation is directly modulated by Xist non-coding RNA [E8.5]

X-chromosome inactivation silences one copy of the X-chromosome in female cells, but a number of genes escapes XCI. We asked whether increased levels of Xist RNA, the molecular actor driving X-inactivation, impacts expression levels of escapees. We tested whether dox-mediated Xist-overexpression affected the expression levels of escapees in extra-embryonic tissues. Overall design: To investigate the effect of Xist overexpression on imprinted X-inactivation in extra-embryonic tissues, we crossed TX/Y males carrying the rtTA transactivator (Xptet/Y; R26rtTA/rtTA or Xptet/Y; R26rtTA/WT) with wild-type JF1 females. Xist RNA overexpression was induced by adding doxycycline to the drinking water of pregnant females for 5 days, from E3.5 to E8.5. RNA was extracted from the collected extra-embryonic tissues of each embryo. Libraries were prepared using the Smart-Seq2 protocol, and allele-specific expression was determined for X-linked genes.