Identify METTL3, 14 and 16-bound transcripts via RIP-seq

To identify the directly bound transcripts of METTL3, METTL14, and METTL16, RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T stablely expressing METTL3, METTL14, and METTL16, respectively. Briefly, HEK293T cells were infected with lentivirus, pmiRNA1-3 x Flag-METTL3, pmiRNA1-3 x Flag-METTL14, and pmiRNA1-3 x Flag-METTL14, to overexpress the three METTL family members with 3 x Flag fused in the N-terminal. Only the GFP-positive cells were used for study and expanded in DMEM medium. The GFP-positive HEK293T cells with overexpression of METTL3, METTL14, and METTL16, were expanded in 150mm dish and collected at 70-80% confluency. The were cross-linked by 0.75 % formaldehyde (F8775, Sigma-Aldrich) with gentle rotation at room temperature for 10 min and the reaction was quenched by 125 nM glycine (final concentration) with shaking at room temperature for 5 min. Then cells were collected, lysed, and subjected to RIP with Flag antibody. Two biological replicates were included to confirm the data quality.