m6A-seq profiling in Mettl3 Ctrl and dTAG13 mESCs [m6Aseq]

dTAG-13 treated METTL3_FKBP12(F36V) is sufficient to functionally deplete m6A methylation, that catalysed by METTL3/14 complex. Overall design: We would like to confirm the functional m6A loss by dTAG-13 treatment in the Mettl3_FKBP12(F36V) cells. C3 and H5 are two independent clones correspondingly. The Female mouse embryonic stem cells expressing dox-inducible Xist were employed to generate this dTAG line because Xist is heavily methylated by m6A so that it can be used as a marker to test the presence or absence of m6A modification. Our western blot analysis revealed that Mettl3_FKBP12(F36V) is sensitive to dTAG-13 treatment and 2 hours are sufficient to deplete the protein. Therefore, cells are either treated with dTAG-13 or not for 2 hours, and then added the doxycycline to induce Xist expression for another 24 hours. Total RNAs from these treated cells were isolated and fragmented with independent Drosophila total RNAs in parallel. All the fragmented RNAs were quantified well by Nanodrop and Qubit, then the equal ms of fragmented mESC RNAs (300ug) were mixed with 30ug of Drosophila total RNAs and subjected to standard m6A-seq. Fragmented mESC total RNA were used input-RNA-seq and used for the conventional m6A-seq analysis.