Nucleotide resolution profiling of m(7)G tRNA modification by TRAC-Seq.
Nucleotide resolution profiling of m(7)G tRNA modification by TRAC-Seq.
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通过 TRAC-Seq 对 m(7)G tRNA 修饰进行核苷酸分辨率分析
DOI：
10.1038/s41596-019-0226-7
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发表时间：
2019-11
期刊：
影响因子：
14.8
通讯作者：
Gregory RI
中科院分区：
文献类型：
--
作者：
Lin S;Liu Q;Jiang YZ;Gregory RI
RNA修饰位点的精确鉴定是研究这种修饰在基因表达调控中的功能作用以及阐明与不同生理过程相关性的关键。tRNA还原和切割测序（TRAC-Seq）是一种基于化学的方法，用于在整个tRNA转录组中以单核苷酸分辨率对tRNA的7-甲基鸟嘌呤（m7G）修饰进行无偏全局映射。m7G TRAC-Seq涉及用脱甲基酶AlkB处理大小选择的（<200 nt）RNA以去除主要的tRNA修饰，然后用硼氢化钠（NaBH 4）还原m7G位点，随后在所得脱碱基位点处苯胺介导的RNA链切割。随后将切割的位点与衔接子连接以构建用于高通量测序的文库。使用生物信息学管道来识别m7 G修饰位点，该管道计算所有tRNA上各个位点的切割评分。与基于抗体的方法不同，例如用于富集甲基化RNA序列的甲基化RNA免疫沉淀和测序（meRIP-Seq），基于化学的方法（包括TRAC-Seq）可以提供修饰位点的核苷酸水平分辨率。与相关的AlkAniline-Seq方法（碱性水解和苯胺裂解测序）相比，TRAC-Seq结合了小RNA选择，AlkB去甲基化和硼氢化钠还原步骤，以实现tRNA中m7G位点的特异性和有效的单核苷酸分辨率分析。m7 G TRAC-Seq方案可以适用于化学切割介导的其他RNA修饰检测。该方案可以在约9天内完成四个生物重复的输入和处理的样品。
Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased global mapping of 7-methylguansine (m7G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m7G TRAC-Seq involves the treatment of size-selected (<200 nt) RNAs with the demethylase AlkB to remove major tRNA modifications, followed by sodium borohydride (NaBH4) reduction of m7G sites and subsequent aniline-mediated cleavage of the RNA chain at the resulting abasic sites. The cleaved sites are subsequently ligated with adaptors for the construction of libraries for high-throughput sequencing. The m7G modification sites are identified using a bioinformatic pipeline that calculates the cleavage scores at individual sites on all tRNAs. Unlike antibody-based methods, such as methylated RNA immunoprecipitation and sequencing (meRIP-Seq) for enrichment of methylated RNA sequences, chemically based approaches, including TRAC-Seq, can provide nucleotide-level resolution of modification sites. Compared to the related method AlkAniline-Seq (alkaline hydrolysis and aniline cleavage sequencing), TRAC-Seq incorporates small RNA selection, AlkB demethylation, and sodium borohydride reduction steps to achieve specific and efficient single-nucleotide resolution profiling of m7G sites in tRNAs. The m7G TRAC-Seq protocol could be adapted to chemical cleavage-mediated detection of other RNA modifications. The protocol can be completed within ~9 d for four biological replicates of input and treated samples.
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影响因子：
14.9
作者：
Kawarada L;Suzuki T;Ohira T;Hirata S;Miyauchi K;Suzuki T
通讯作者：
Suzuki T
DOI：
10.1093/bioinformatics/btq033
发表时间：
2010-03-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
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作者：
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通讯作者：
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48
作者：
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通讯作者：
Salzberg, Steven L.
模因套件：用于发现和搜索的工具。
影响因子：
14.9
作者：
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通讯作者：
Noble WS
影响因子：
12.3
作者：
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通讯作者：
Alkuraya FS