Accuprime supermix 1 enzyme

Single-nucleotide-resolution mapping of m⁶A of A498 cells was carried out according to previously published studies (Chen et al., 2015 (link); Linder et al., 2015 (link)) with some modifications. Briefly, the mRNA of A498 cells were purified by Dynabeads mRNA Purification Kit (Life Technologies) and fragmented to about 100 nt by the fragmentation reagent (Life Technologies). 2 μg of fragmented mRNAs were incubated with 5 μg of anti-m6A antibody (Abcam) in 300 μl immunoprecipitation buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40) at 4°C for 2 h. The mixture was then irradiated three times with 0.15 Jcm^–2 at 254 nm by a CL-1000 Ultraviolet Crosslinker (UVP), and then incubated with Dynabeads Protein A (Life Technologies) at 4°C for 2 h. After washing, end-repair and linker ligation, the enriched RNA were isolated from the beads by proteinase K digestion, and extracted by phenol–chloroform. Purified RNAs were reverse transcribed by Superscript III reverse transcriptase (Life Technologies). The cDNA from last step was with size selection on a 6% TBE-Urea gel (Life Technologies), and circularization and re-linearization by CircLigase II (Epicenter) and BamHI (NEB), respectively. Then the cDNA was amplified by AccuPrime SuperMix 1 enzyme (Life Technologies) for 20 cycles and sequenced by Illumina HiSeq X Ten according to the manufacturer’s instructions.