Proximity-CLIP and Expedited Non-Radioactive Library Preparation of Small RNA Footprints for Next-Generation Sequencing.
Proximity-CLIP and Expedited Non-Radioactive Library Preparation of Small RNA Footprints for Next-Generation Sequencing.
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DOI：
10.1002/cpmb.120
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发表时间：
2020-06-01
影响因子：
--
通讯作者：
Hafner, Markus
中科院分区：
文献类型：
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作者：
Anastasakis, Dimitrios;Benhalevy, Daniel;Hafner, Markus
在它们的生命周期过程中，大多数RNA在几个细胞环境之间移动，在那里它们与不同的RNA结合蛋白（RBP）结合。相反，RBP的很大一部分存在于一个以上的细胞区室中，在那里它们可以与离散的RNA相互作用，甚至发挥不同的生物学作用。邻近-CLIP结合蛋白质的邻近生物素化与可光活化的核糖核苷增强的蛋白质-RNA交联，以在任何给定的亚细胞区室中同时分析蛋白质组，包括RBP和RBP结合的转录组。在这里，我们提供了一个详细的实验方案的接近CLIP沿着与简化的非放射性，小RNA cDNA文库制备方案。2020年美国政府。基本方案1：基本方案2：细胞提取、链霉亲和素亲和纯化和珠上胰蛋白酶消化基本方案3：RNA足迹cDNA文库制备支持方案：从完整RNA制备RNA-seq文库。
During the course of their life cycle, most RNAs move between several cellular environments where they associate with different RNA binding proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity-CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA crosslinking to simultaneously profile the proteome, including RBPs and the RBP-bound transcriptome, in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity-CLIP along with a simplified non-radioactive, small-RNA cDNA library preparation protocol. Published 2020 U.S. Government. Basic Protocol 1: Cell culture, 4SU labeling, proximity biotinylation, and crosslinking Basic Protocol 2: Cell extraction, streptavidin affinity purification, and on-beads trypsinization Basic Protocol 3: RNA footprints cDNA library preparation Support Protocol: Preparation of RNA-seq libraries from intact RNA.