title: NextSeq 500 paired end sequencing; RNAseq in Dppa2 knockout or wildtype TX1072 ESC during and after Xist over-expression
design: RNAseq in Dppa2 knockout or wildtype TX1072 ESC during and after Xist over-expression
library_construction_protocol: TX1072 are XX mouse embryonic cell line mixed Cast/BL6 (Shultz et al., 2014) that carry a DOX inducible Xist on the Bl6 allele. To induce XCI culture ES cells with 100 ug/ml of DOX for 6 days in DMEM (Sigma), 15% FBS (Gibco), 1% Penicillin streptavidin, 0.1 mM _-mercaptoethanol, 1000 U/ml (LIF), CHIR99021 (3 _M), PD0325901 (1 _M). To induce XCR wash out DOX (D-wo) and harvest samples for analysis at the following time-series: 1d D-wo, 3d D-wo and 7d D-wo. total RNA was extracted using the Monarch Total RNA Miniprep Kit (NEB) (#T2010S) Libraries for RNA-seq by enrichment of PolyA+ mRNA were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490)