Quantifying m6A Levels in LUAD Cells

The MeRIP assay was used to determine the m6A levels of the target gene ADIRF in A549 and H157 cells.^{33 (link)} Total RNA was isolated from A549 and H157 cells. Stable high-expressing ALKBH5 and LUAD cells transfected with a vacant vector were used as controls (A549/vector, H157/vector). The obtained mRNA was purified using DNase (Sigma) and fragmented into ~ 100 nucleotides. m6A primary antibody (10 μg, Abcam, ab151230) linked to Magna ChIP protein/G magnetic beads was co-immunoprecipitated with the fragmented mRNA for 2 h at 4°C according to the protocols of the Magna MeRIP™ m6A kit (17–10499, Millipore, Germany). m6A RNA elution and purification were performed using an eluent containing N6-methyladenosine 5’-monophosphate sodium salt (twice, 1 h), followed by overnight precipitation in ethanol (−80°C). qRT-PCR was then performed on a 7500 Fast RT-PCR instrument (Applied Biosystems) to analyze the mRNA levels of m6A sites.