Nucleotide resolution profiling of m3C RNA modification by HAC-seq. Nucleotide resolution profiling of m3C RNA modification by HAC-seq
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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.
本研究报道了一种针对N3-甲基胞嘧啶（m3C）的高通量测序技术——肼-苯胺裂解测序（Hydrazine-Aniline Cleavage sequencing，HAC-seq），可在单核苷酸分辨率下绘制m3C甲基化组图谱。我们将HAC-seq应用于分析MCF7细胞中经核糖体RNA去除后的总RNA。研究发现，转运RNA（transfer RNA，tRNA）是主要的m3C修饰RNA类型；在MCF7细胞中，未检测到信使RNA（messenger RNA，mRNA）或其他非编码RNA存在与tRNA相当水平的m3C修饰。 整体实验设计：HAC-seq设置两组生物学重复。经核糖体RNA去除后，将去核糖体RNA总RNA进行随机片段化并末端修复。将片段化RNA置于含3M氯化钠的10%肼溶液中处理4小时，随后通过苯胺裂解缓冲液在m3C修饰位点处进行裂解（即HAC处理）。文库制备前，需使用AlkB酶对RNA进行处理。实验同时设置两组对照：未处理对照组（CTRL）仅进行AlkB去甲基化处理，不施加肼处理与苯胺裂解；DM-HAC组则先经AlkB去甲基化，再依次进行肼处理与苯胺裂解。
