RIPiT-Seq: A tandem immunoprecipitation approach to reveal global binding landscape of multisubunit ribonucleoproteins.
RIPiT-Seq: A tandem immunoprecipitation approach to reveal global binding landscape of multisubunit ribonucleoproteins.
复制标题
DOI：
10.1016/bs.mie.2021.03.019
复制
发表时间：
2021
影响因子：
--
通讯作者：
Singh G
中科院分区：
文献类型：
--
作者：
Yi Z;Singh G
RNA结合蛋白（RBP）调节RNA代谢的各个方面。识别RBP结合的RNA靶点的能力对于理解RBP功能至关重要。虽然强大的技术可用于以高分辨率鉴定单个RBP的结合位点，但解开多组分核糖核蛋白（RNP）的结合位点仍然具有挑战性，其中多个RBP或蛋白质协同作用以结合靶RNA。为了填补这一空白，我们之前已经开发了RNA免疫沉淀串联，然后进行高通量测序（RIPiT-seq），通过顺序免疫沉淀来自相同RNP的两种蛋白质并对共纯化RNA足迹进行测序来表征组成不同的RNP复合物的RNA靶标。在这里，我们为RIPiT-seq提供了更新和改进的协议。在该方案中，我们使用CRISPR-Cas9将亲和标签引入到感兴趣的内源蛋白质中，以捕获RNP复合物的更具代表性的状态。我们提出了一种用于高通量测序的文库制备的修改方案，以便它专门使用标准分子生物学实验室中可用的设备和试剂。这种更新的定制文库制备方案与Illumina测序平台的商业PCR多重系统兼容，可同时分析大量样品并具有成本效益。
RNA-binding proteins (RBPs) regulate all aspects of RNA metabolism. The ability to identify RNA targets bound by RBPs is critical for understanding RBP function. While powerful techniques are available to identify binding sites of individual RBPs at high resolution, it remains challenging to unravel binding sites of multicomponent ribonucleoproteins (RNPs) where multiple RBPs or proteins function cooperatively to bind to target RNAs. To fill this gap, we have previously developed RNA Immunoprecipitation in Tandem followed by high-throughput sequencing (RIPiT-seq) to characterize RNA targets of compositionally distinct RNP complexes by sequentially immunoprecipitating two proteins from the same RNP and sequencing the co-purifying RNA footprints. Here, we provide an updated and improved protocol for RIPiT-seq. In this protocol, we have used CRISPR-Cas9 to introduce affinity tag to endogenous protein of interest to capture a more representative state of an RNP complex. We present a modified protocol for library preparation for high-throughput sequencing so that it exclusively uses equipment and reagents available in a standard molecular biology lab. This updated custom library preparation protocol is compatible with commercial PCR multiplexing systems for Illumina sequencing platform for simultaneous and cost-effective analysis of large number of samples.
登录
查看更多内容
影响因子：
4.6
作者：
Ramlee MK;Yan T;Cheung AM;Chuah CT;Li S
通讯作者：
Li S
影响因子：
16.6
作者：
Singh G;Pratt G;Yeo GW;Moore MJ
通讯作者：
Moore MJ
影响因子：
3.7
作者：
Rivera-Torres N;Strouse B;Bialk P;Niamat RA;Kmiec EB
通讯作者：
Kmiec EB
DOI：
10.1007/978-1-4939-7339-2_1
发表时间：
2018-01-01
期刊：
ARGONAUTE PROTEINS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Gangras, Pooja;Dayeh, Daniel M.;Singh, Guramrit
通讯作者：
Singh, Guramrit
影响因子：
5.8
作者：
Kucukural, Alper;Oezadam, Hakan;Cenik, Can
通讯作者：
Cenik, Can