RIKEN Omics Science Center, RIKEN Yokohama Institute, Yokohama, Japan.
Nat Protoc. 2012 Feb 23;7(3):542-61. doi: 10.1038/nprot.2012.005.
Cap-analysis gene expression (CAGE) provides accurate high-throughput measurement of RNA expression. CAGE allows mapping of all the initiation sites of both capped coding and noncoding RNAs. In addition, transcriptional start sites within promoters are characterized at single-nucleotide resolution. The latter allows the regulatory inputs driving gene expression to be studied, which in turn enables the construction of transcriptional networks. Here we provide an optimized protocol for the construction of CAGE libraries on the basis of the preparation of 27-nt-long tags corresponding to initial bases at the 5' ends of capped RNAs. We have optimized the methods using simple steps based on filtration, which altogether takes 4 d to complete. The CAGE tags can be readily sequenced with Illumina sequencers, and upon modification they are also amenable to sequencing using other platforms.
帽状分析基因表达（CAGE）可提供 RNA 表达的高通量精确测量。CAGE 允许对所有带帽编码和非编码 RNA 的起始位点进行作图。此外，还可以在单个核苷酸分辨率下对启动子内的转录起始位点进行表征。后者允许研究驱动基因表达的调节输入，进而构建转录网络。在这里，我们提供了一种基于制备对应于 capped RNA 5' 端初始碱基的 27nt 长标签的优化方案，用于构建 CAGE 文库。我们使用基于过滤的简单步骤对方法进行了优化，总共需要 4 天时间完成。CAGE 标签可以很容易地使用 Illumina 测序仪进行测序，并且经过修饰后，它们也可以使用其他平台进行测序。
