The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation

Input mRNAs and ribosome protected fragments (RPFs) from wildtype and Mettl5 knockout (KO) mESC cells were analyzed by TruSeq Ribo Profile (Illumina) library preparation and high throughput sequencing Overall design: The mESCs were cultured in 2i/lif medium. The 2i/lif medium contained N2B27 medium with 2i (PD0325901:1µM, Selleck; CHIR99021: 3µM, Selleck) and leukemia inhibitory factor lif (1,000U/ml, Millipore). N2B27 medium consisted of 50% DMEM/F12(GIBCO, #10565-018),50%Neurobasal medium (GIBCO, #21103-049) supplemented with 1xN2 (GIBCO, #17502048), 1xB27 (GIBCO, #17504044), 1xGlutamax (GIBCO, #35050061),1mM NEAA (GIBCO, #11140050),1xSodium Pyruvate (GIBCO, #11360070) and 0.1mM 2-mercaptoethanol (Sigma-Aldrich, #M3148). Input mRNAs and RPFs were isolated, ribosomal RNAs were depleted and cDNA libraries were prepared according to the TruSeq Ribo Profile pipeline (Illumina)