Identify Flag-bound transcripts via RIP-seq

To identify the directly bound transcripts of Flag antibody (the backgroud control for our METTL16 RIP-seq), RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T. Briefly, HEK293T cells were infected with pmiRNA1-empty vector. Only the GFP-positive cells were used for study and expanded in DMEM medium. Overall design: The GFP-positive HEK293T cells 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.