The libraries were sequenced on an Illumina HiseqXten-PE150, at a depth of ~20 million reads per sample. The reads were mapped to the human reference genome (hg38) by STAR software (Version 2.5.1); annotation from GENCODE version V30 was used. After removing duplication, variants were identified by GATK (Version; MuTect2 and HaplotypeCaller). For MuTect2 method, variants were filtered with FilterMutectCalls. For HaplotypeCaller method, variants were first filtered with QD (Quality by Depth) <2, then all variants were verified and quantified by bam-readcount with parameters -q 20 -b 30. The depth for a given edit should be at least 10x and these edits were required to have at least 99% of reads supporting the reference allele in the wild-type samples. Finally, only A-to-G edits in transcribed strand were considered for subsequent analysis. Motif or sequence logo was analyzed by WebLogo (v3.6.0) for RNA edits. The downloaded data subjected to RNA off-target analysis from four published papers were listed in Source Data for Supplementary Figures. Detailed information for called mutations was provided in in a Source Data file (Source Data for called mutations from RNA-seq data).

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