Determine the methyltransferase activities of METTL3, METTL14, and METTL16 via MeRIP-seq (m6A-seq)

All the three enzymes, METTL3, METTL14, and METTL16, belong to methyltransferase like (METTL) family and possess the ability to deposit N6-methyladenosine (m6A) in mRNA. Via immunofluorescence staining and wertern blotting, we discovered either METTL3 or METTL14 mainly localize in nuclear, but METTL16 localizes in both cytosol and nuclei. To compare the m6A methyltransferase activities of the three m6A 'writers' and better understand their differences, MeRIP-seq (m6A-seq) was conducted with poly(A) RNAs isolaed from HEK293T cells with METTL3, 14 and 16 knockout. Overall design: The HEK293T-Cas9 single clone cells were infected with sgMETTL3-3, sgMETTL14-3, sgMETTL16-3, and sgNS lentivirus to induce knockout of the m6A writers. The positive-infected cells were selected with 10ug/ml blasticidin (for Cas9) and 100ug/ml hygromycin (for sgRNAs) for 10 days. For each group, we included 2 biological replicates. The the cells were collected and total RNA were isolated from the whole cells via Qiazol. The poly(A) RNAs were further enriched from the total RNA and then subjected to m6A IP.