修订后的iCLIP-seq协议,用于分析活细胞中个别核酸分辨率的RNA-蛋白相互作用位点
Syed Nabeel-Shah1,2, Jack F Greenblatt1,2
1Department of Molecular Genetics, University of Toronto, Toronto, M5S 1A8, Canada.
Bio-protocol
|June 16, 2023
概括
我们开发了iCLIP-1.5,这是一种精细的方法,用于识别单核酸分辨率下与RNA的RNA结合蛋白 (RBP) 相互作用. 这种增强的技术提高了效率和覆盖率,即使是低输入样本,有助于RBP研究.
科学领域:
- 分子生物学分子生物学
- 基因组学就是基因组学.
- 在RNA生物学,RNA生物学.
背景情况:
- 单个核酸分辨率紫外线交叉链接和免疫沉,其次是高通量测序 (iCLIP-seq),对于识别RNA结合蛋白 (RBP) 结合部位至关重要.
- 现有的CLIP变体如iCLIP2和eCLIP旨在提高效率和简化协议.
- 转录因子SP1已经涉及到通过直接RNA结合调节替代裂变和多化.
研究的目的:
- 为了提高效率和稳定性,提出修订后的iCLIP-seq协议,称为iCLIP-1.5.
- 为了确定SP1的RNA结合部位和分裂和多基化复合物的子单元.
- 为那些难以CLIP的蛋白质提供替代方法.
主要方法:
- 一种经过修改的iCLIP方法,采用eCLIP的特性,优化cDNA循环化.
- 对iCLIP-1.5协议进行详细的逐步程序.
- 该协议的应用用于确定SP1,CFIm25,CPSF7,CPSF100,CPSF2和Fip1.1的绑定位点.
主要成果:
- 与以前的方法相比,ICLIP-1.5协议显示了更高的效率和稳定性.
- 实现了更高的测序覆盖率,特别是对于低输入样本.
- 在核酸分辨率下获得了关于RBP-RNA相互作用的精确位置和定量信息.
结论:
- 该iCLIP-1.5协议是一个高效和强大的方法,用于绘制全基因组的RBP结合位点.
- 这种技术有助于识别RBP序列动机,并对蛋白质-RNA相互作用进行定量分析.
- 修订后的协议通过提供详细的RBP-RNA相互作用数据,推进了对转录后调节途径的研究.
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