Gene-specific m6A qPCR

Thiamethoxam resistant and susceptible mRNAs were subject to Methylated RNA Immunoprecipitation (MeRIP) analysis using the Magna MeRIP m⁶A kit (17-10499, Millipore) to profile of m⁶A RNA methylation sites. CYP4C64-specific m⁶A qPCR was used to determine m⁶A abundance across transcripts of this gene. Briefly, 30μg of total RNA was sheared to about 100nt in length by metal-ion-induced fragmentation, then purified and incubated with anti-m⁶A antibody-conjugated beads in 100μl 1×immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C for 2 hours. Methylated RNA was immunoprecipitated with beads, eluted by competition with free m⁶A, and recovered with elution buffer, and converted to cDNA (Taraka Biotech), further analysed by qPCR along with MeRIPed RNA (TIANGEN). The relative enrichment of m⁶A in each sample was calculated by normalizing to a reference gene EF1α (RPL29 cannot determine in the MeRIPed RNA). Each group was repeated in triplicate.