m6A-seq profiling in WT, WtapKO and Mettl3KO mESCs

By m6A-seq, we examined the m6A pattern on Xist, which immediately after the A-repeat region. The cell line, integrating multiple copy of Xist on chr3, with m6A core machinery knockout (Wtap and Mettl3) were generated. We also profiled m6A-seq in transgenic Xist line, and observed a global reduction of m6A level, validated by m6A-LC-MS/MS. Unexpectedly, we haven't observed reduced m6A level on Xist m6A region downstream of A-repeat in transgenic Xist line. Overall design: M6A profiling in WT, WtapKO, and Mettl3KO male mESC cells, in which Xist RNA was randomly integrated into Chr3 under the TetOn promoter. After dox treatment, Xist expressed from chr3 locus and leads to chr3 inactivation. We are aiming to understand how m6A pattern was on Xist and how m6A modification on Xist affect Xist-mediated chromosome silencing.