PDF(Pubmed)
Abstract：
The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3\' end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.We provide a step-by-step instruction for time-resolved mRNA 3\' end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.
摘要：
mRNAs衰变的不同速率与转录变化紧密协调，以在发育和疾病期间塑造基因表达。但是目前可用的RNA测序方法缺乏时间信息来确定RNA生物发生的相对贡献，加工和周转以建立稳态基因表达谱。这里,我们描述了一种方案，该方案将代谢RNA标记与通过4-硫尿苷的巯基连接烷基化的化学核苷转化相结合，以确定培养细胞中的RNA稳定性（SLAMseq）。当与具有成本效益的mRNA3'端测序方法相结合时，SLAMseq使用非靶向或靶向cDNA文库制备方案以全局和转录特异性方式确定聚腺苷酸化转录物的半衰期。我们提供了时间分辨mRNA3'末端测序的分步说明，它增强了传统的RNA-seq方法，以获得研究控制基因表达的分子原理所需的时间分辨率。
