Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5'-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing.
Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5'-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing.
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RNA 交联免疫沉淀 (CLIP) 标签的分离、5-接头连接、逆转录聚合酶链反应 (RT-PCR) 扩增和测序。
DOI：
10.1101/pdb.prot097972
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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
该方案描述了通过交联蛋白的蛋白酶 K 消化、向 RNA 标签添加 5' 接头以及通过转录聚合酶链式反应 (RT-PCR) 扩增产物来纯化 RNA 交联免疫沉淀 (CLIP) 标签。使用该方案增加了另一个重要的纯化步骤：确定 PCR 产物的大小，以富集源自最初与所需 RNABP 交联的 RNA 的产物。最后，描述了 PCR 产物的测序。对“CLIPed”RNA 的 PCR 产物进行测序有两种策略。低通量测序涉及 PCR 产物的克隆、常规小量制备和测序。这可以使用用于 PCR 产物 A 加尾和 TA 克隆的标准方案对此处生成的 PCR 产物进行。在分析少量克隆时，这可能是一个有价值的策略。一般来说，特别是考虑到成本下降，高通量测序是对 CLIPed RNA 产物进行测序的首选方法。该协议描述了一种使用适合在 Illumina 的 Solexa 平台上使用的引物重新扩增 PCR 产物的方法。尽管该方案特定于 Illumina 深度测序平台，但可使用类似的初始 PCR 产物再扩增方案将平台特定序列添加到 PCR 扩增 DNA 的末端。
This protocol describes purification of RNA cross-linking immunoprecipitation (CLIP) tags by proteinase K digestion of the cross-linked protein, addition of a 5′ linker to the RNA tags, and amplification of the product by transcription-polymerase chain reaction (RT-PCR). Use of this protocol adds another important purification step: sizing of the PCR products to enrich for those derived from RNA originally cross-linked to the desired RNABP. Finally, sequencing of the PCR products is described. There are two strategies for sequencing the PCR products of “CLIPed” RNA. Low-throughput sequencing involves cloning of PCR products, conventional minipreps, and sequencing. This can be performed on the PCR products generated here using standard protocols for A-tailing the PCR product and TA-cloning. This may be a worthwhile strategy when analyzing a small number of clones. In general, particularly in light of falling costs, high-throughput sequencing is the preferred method for sequencing the products of CLIPed RNA. This protocol describes a method for reamplifying PCR products with primers suitable for use on Illumina’s Solexa platform. Although this protocol is specific to the Illumina deep-sequencing platform, similar schemes for reamplification of the initial PCR products can be used to add platform-specific sequences to the termini of the PCR-amplified DNA.