3'-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).
3'-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).
复制标题
通过 SDS-PAGE 进行 3-接头连接和大小选择，用于交联免疫沉淀 (CLIP)。
DOI：
10.1101/pdb.prot097964
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发表时间：
2018
影响因子：
--
通讯作者：
Darnell,RobertB
中科院分区：
文献类型：
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作者：
Darnell,JenniferC;Mele,Aldo;Hung,KaYingSharon;Darnell,RobertB
该方案描述了通过变性聚丙烯酰胺凝胶电泳进行交联免疫沉淀 (CLIP) 的 RNA 接头的纯化。纯化是必要的，因为如果 3' 连接子失去嘌呤霉素封闭基团，则在 3' 连接子连接反应期间将发生 3' 连接子的多联化。此外，截短的接头使测序结果的生物信息处理变得比需要的更加困难。此外，该协议描述了用碱性磷酸酶处理 CLIP 的共免疫沉淀 RNA 标签，以去除 RNase 消化后剩余的 3' 磷酸盐。去磷酸化可防止 RNA 在随后与接头连接过程中发生分子内环化。纯化的 RNA 接头在其 3' 末端用嘌呤霉素封闭以防止接头-接头多聚化，然后连接到 RNA 标签的 3' 末端。通过执行连接来去除游离接头，同时 RNABP：RNA 复合物通过抗体与 Protein A Dynabeads 结合，从而实现彻底清洗和接头去除。通过 SDS-PAGE 以及将选定大小的 RNABP：RNA 复合物转移至硝酸纤维素上，实现额外的纯化。
This protocol describes the purification by denaturing polyacrylamide gel electrophoresis of RNA linkers for cross-linking immunoprecipitation (CLIP). Purification is necessary because if the 3′ linker loses the puromycin blocking group, concatemerization of the 3′ linker will occur during the 3′ linker ligation reaction. In addition, truncated linkers make bioinformatic processing of the sequencing results more difficult than it need be. Additionally, this protocol describes the treatment of coimmunoprecipitated RNA tags for CLIP with alkaline phosphatase to remove the 3′ phosphate remaining after RNase digestion. Dephosphorylation prevents intramolecular circularization of RNA during subsequent ligation to the linker. The purified RNA linker, blocked with puromycin at its 3′ end to prevent linker–linker multimerization, is then ligated to the 3′ end of the RNA tag. Removal of free linker is accomplished by performing the ligation while the RNABP: RNA complex is associated, via antibody, to protein A Dynabeads, allowing thorough washing and linker removal. Additional purification is achieved by SDS-PAGE and transfer of the size-selected RNABP: RNA complexes to nitrocellulose.