核糖核酸
生物
抄写(语言学)
增强子
RNA聚合酶Ⅱ
计算生物学
RNA聚合酶
遗传学
基因
基因组
深度测序
发起人
基因表达
语言学
哲学
作者
Petros Tzerpos,Bence Dániel,László Nagy
出处
期刊:Methods in molecular biology
日期:2021-01-01
卷期号:: 25-39
被引量:4
标识
DOI:10.1007/978-1-0716-1597-3_2
摘要
Post-transcriptional processing strongly affects the stability and the relative quantification of RNA molecules, so that steady-state levels of mature RNA, such as mRNAs, rarely reflect accurately the rate of in situ transcription in nuclei by RNA polymerases (RNAPs). The "Global Run-on Sequencing (GRO-Seq)" method, developed in 2008, combines the nuclear run-on assay with next-generation deep sequencing to detect nascent RNA levels to annotate the positions, the relative levels and the orientation of transcriptionally engaged RNA polymerase II (RNAPII) molecules genome-wide. Thus, GRO-Seq is a powerful method to infer mechanistic insights into the multiple levels of transcriptional regulation such as promoter-proximal pausing of RNAP, bidirectional transcription, and enhancer activity. Here, we describe a protocol for mammalian cells that can reliably detect low abundant nascent RNA from both coding and noncoding genomic regions. This protocol can easily be adapted for most mammalian cells to define the transcriptionally active regions of the genome and to measure dynamic transcriptional responses with high sensitivity upon external stimuli.
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