Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland.
Curr Protoc Mol Biol. 2020 Jun;131(1):e120. doi: 10.1002/cpmb.120.
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.
在其生命周期过程中，大多数 RNA 在几种细胞环境之间移动，在这些环境中，它们与不同的 RNA 结合蛋白 (RBP) 结合。反过来，相当一部分 RBP 存在于不止一个细胞区室中，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 将蛋白质的邻近生物素化与光活化核核苷增强的蛋白质-RNA 交联结合起来，可同时在任何特定的亚细胞区室中对蛋白质组（包括 RBP 和 RBP 结合的转录组）进行分析。本文提供了详细的邻近 CLIP 实验方案，以及简化的非放射性、小 RNA cDNA 文库制备方案。出版 2020 年美国政府。基本方案 1：细胞培养、4SU 标记、邻近生物素化和交联基本方案 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：完整 RNA 的 RNA-seq 文库制备。
