m6A-REF-seq protocol was used to identify m6A sites in the different tissues of human (brain, liver, and kidney), mouse (brain, liver, heart, testis, and kidney), and rat (brain, liver, and kidney). The one-to-one orthologous genes in pairwise species of human (GRCh37.p13), mouse (GRCm38.p6), and rat (Rnor_6.0) were downloaded from Ensembl (Ensembl genes 94). A total of 13,117 one-to-one orthologous genes among three species were retained for downstream analyses. Protein sequences of 13,117 orthologs in human, mouse, and rat were aligned by MUSCLE (37) and then converted to corresponding coding sequence alignments using PAL2NAL (38). The shared m6A-modified genes and share m6A sites among species were calculated and tested for significances based on the well-aligned orthologous coding-region sequences as mentioned above. The average pairwise distances among closely m6A sites in three species were calculated and compared with random control sample. The control sample was randomly picked for the same number of ACA motifs as the number of observed m6A for 1000 times. Analyses of conservation score were conducted by using PhastCons scores of human (hg19), mouse (mm10), and rat (rn6) downloaded from University of California, Santa Cruz (UCSC).

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