Nucleotide resolution profiling of m3C RNA modification by HAC-seq

We report a m3C-specific high-throughput sequencing techinque, Hydrazine-Aniline Cleavage sequencing (HAC-seq) to profile the m3C methylome at single-nucleotide resolution. We apply HAC-seq to analyze ribosomal RNA-depleted total RNA from MCF7 cells. We find that tRNA are the predominant m3C-modified RNA species. We find no evidence of m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNA in MCF7 cells. Overall design: HAC-seq was performed in biological duplicates. After ribosomal RNA depletion, ribominus total RNA were randomly fragmented and end-repaired. The fragmented RNAs were treated with 10% hydrazine with 3M NaCl for 4h and then cleaved by aniline cleavage buffer at the m3C modification sites (HAC). Then RNAs were treated with AlkB before library preparation. Two controls were performed at the same time. Untreated control (CTRL) was carried out with AlkB demethylation but without hydrazine and aniline cleavage. DM-HAC was performed by treating the AlkB-demethylated RNA with hydrazine followed by aniline cleavage.