Development and Validation of BruChase-Seq and BrUV-Seq

描述（由申请人提供）：创新的高通量全基因组方法和技术（如RNA-Seq和ChIP-Seq）的发展使得鉴定和分析所有编码和非编码RNA以及绘制表观遗传标记和转录因子结合位点成为可能。然而，RNA-Seq通常使用从细胞或组织分离的总RNA进行，并且这种分析将产生RNA的稳态水平的特征，但不会告知特定基因表达改变是否是由于转录改变和/或RNA稳定性改变。最近，已经开发了许多不同的方法来估计基因组序列的新生转录，例如GRO-Seq、NET-Seq和通过代谢标记和分离新生RNA。在R 01申请中，我们将进一步开发和验证两种方法，我们认为这两种方法为现有技术增加了重要的新功能，用于全面和高通量探索人类细胞中的基因表达特征。BruChase-Seq基于溴尿苷脉冲追踪标记结合深度测序，直接测量所有初级和成熟mRNA和非编码RNA的合成和降解动力学，并确定所有内含子序列的剪接动力学。BrUV-Seq在BrU标记之前通过UV光引入随机转录阻断损伤，鉴定转录起始位点（TSS）、推定的增强子元件，并且通过稳定如果完全转录则可能不稳定的转录物，从而允许其更灵敏的检测。这些进展中的每一个都有可能通过对已知基因进行额外的功能评估来大大扩展当前的基因组注释，显示基因的各个部分如何独立地起作用，并识别隐藏的基因间元件。

DESCRIPTION (provided by applicant): The development of innovative high throughput genome-wide methodologies and technologies such as RNA-Seq and ChIP-Seq have made it possible to identify and analyze all coding and non-coding RNAs as well as map epigenetic marks and transcription factor binding sites. However, RNA-Seq is typically performed using total RNA isolated from cells or tissues and such analysis will generate signatures of steady-state levels of RNA but will not inform on whether particular gene expression alterations are due to altered transcription and/or altered RNA stability. Recently, a number of different approaches have been developed to estimate nascent transcription of genomic sequences such as GRO-Seq, NET-Seq and by metabolic labeling and isolated of nascent RNA. We will in this R01 application further develop and validate two approaches that we believe add important new capabilities to existing techniques for the comprehensive and high throughput exploration of gene expression signatures in human cells. BruChase-Seq is based on bromouridine pulse-chase labeling coupled to deep sequencing directly measuring the kinetics of synthesis and degradation of all primary and mature mRNAs and non-coding RNAs as well as determines splicing kinetics of all intron sequences. BrUV-Seq introduces random transcription-blocking lesions by UV light prior to BrU-labeling, identifying transcription start sites (TSS), putative enhancer elements and by stabilizing transcripts that might be labile if completely transcribed allowing their more sensitive detection. Each of these advances has the potential to greatly expand current genomic annotations by assigning additional functional assessments to known genes, showing how the parts of genes act independently, and identifying cryptic intergenic elements.