Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq
Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq
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DOI：
10.1038/nprot.2015.064
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发表时间：
2015-07-01
期刊：
影响因子：
14.8
通讯作者：
Assmann, Sarah M.
中科院分区：
文献类型：
--
作者：
Ding, Yiliang;Kwok, Chun Kit;Assmann, Sarah M.
Structure-seq是一种高通量和定量的方法，它以单核苷酸分辨率提供有关RNARNARNA结构的全基因组信息。Structure-seq可以在体内和体外进行，以研究RNARNARNA结构-功能关系，基因表达的RNARNARNA调节和RNARNARNA加工。Structure-seq可以由经验丰富的分子生物学家进行，并对生物信息学有基本的了解。Structure-seq从单次命中动力学条件下的化学RNARNARNA结构探测开始。某些化学修饰，例如，通过硫酸二甲酯使未配对的腺嘌呤和胞嘧啶残基的沃森-克里克面甲基化，导致逆转录终止。然后使用随机六聚体引物对修饰的RNARNARNA进行逆转录，这使3'端偏倚最小化;逆转录继续进行，直到它被化学修饰的残基阻断。通过基于接头的PCRPCR扩增所得cDNA，并进行高通量测序，随后允许在全基因组范围内检索结构信息。与仅提供单个转录本信息的经典方法相比，单个结构测序实验在类似于1个月的时间内提供了数万个RNARNARNA结构的信息。虽然这里描述的程序是在体内拟南芥幼苗，结构测序是广泛适用的，从而开辟了新的途径，探索RNARNARNA结构功能的关系，在活的生物体。
Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNARNARNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNARNARNA structure-function relationships, RNARNARNA regulation of gene expression and RNARNARNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNARNARNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNARNARNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3' end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNANAs are amplified by adapter-based PCRPCRPCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNARNARNA structures in similar to 1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNARNARNA structure-function relationships in living organisms.