The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify the Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation Of the Mammalian Genome (FANTOM). These datasets constitute open resource for TSS annotations and gene expression analysis. Here we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE (LQ-ssCAGE), which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.

中文翻译：

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低量单链CAGE（LQ-ssCAGE）定位调控增强子和启动子

基因表达的上限分析（CAGE）是一种强大的方法，可在测量转录本表达水平的同时，识别加帽的RNA的转录起始位点（TSS）。CAGE允许在所有活性启动子和增强子上以单核苷酸分辨率作图。多年来，大型CAGE数据集已经从各个实验室和财团获得，包括DNA元素百科全书（ENCODE）和哺乳动物基因组功能注释（FANTOM）。这些数据集构成用于TSS注释和基因表达分析的开放资源。在这里，我们为称为低数量（LQ）单链（ss）CAGE（LQ-ssCAGE）的最新CAGE方法提供了一种实验方案，该协议可实现具有成本效益的少量RNA样品分析。LQ-ssCAGE对于从小体积培养的细胞衍生的样品，细胞隔室（如核RNA）或发育阶段的样品特别有用。我们通过从50 ng THP-1细胞提取的RNA中构建240个LQ-ssCAGE文库，证明了该方法的可重复性和有效性，并发现了低表达的新型增强子和启动子衍生的lncRNA。