Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
STAR Protoc. 2021 Sep 16;2(4):100819. doi: 10.1016/j.xpro.2021.100819. eCollection 2021 Dec 17.
The isolation of protein-RNA complexes in the "denaturing cross-linked RNA immunoprecipitation" (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs. For complete details on the use and execution of this protocol, please refer to Rosenberg et al. (2017).
“变性交联 RNA 免疫沉淀”（dCLIP）方案中蛋白质 - RNA 复合物的分离基于生物素标记感兴趣的蛋白质、在体内用 UV 交联 RNA 与蛋白质、RNase 保护分析，以及在变性条件下在链霉亲和素柱上分离 RNA-蛋白质复合物。由于传统的基于抗体的 CLIP 测定法难以应用于多梳复合物，因此 dCLIP 已成功应用，并从其中产生小 RNA 足迹，可进行从头基序分析以鉴定 RNA 结合基序。有关该方案使用和执行的完整详细信息，请参阅 Rosenberg 等人（2017 年）。
