Highlights d m 6 A and m 6 Am methylomes of brain tissues are highly specific d m 6 A and m 6 Am are partially correlated with their writers and erasers d SNPs are enriched in m 6 A-containing regions d Species rather than tissue type is the primary determinant of methylome

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High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

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N6,2′-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. Yet, a specific and sensitive tool to directly map transcriptome-wide m6Am is lacking. Here, we report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5′-UTR N6-methyladenosine (m6A) and enables the identification of m6Am at single-base resolution and 5′-UTR m6A in the human transcriptome. Using m6Am-seq, we also find that m6Am and 5′-UTR m6A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. Collectively, m6Am-seq reveals the high-confidence m6Am and 5′-UTR m6A methylome and provides a robust tool for functional studies of the two epitranscriptomic marks.