Rapid construction of parallel analysis of RNA end (PARE) libraries for Illumina sequencing
Rapid construction of parallel analysis of RNA end (PARE) libraries for Illumina sequencing
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DOI：
10.1016/j.ymeth.2013.06.025
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发表时间：
2014-05-01
期刊：
影响因子：
4.8
通讯作者：
Meyers, Blake C.
中科院分区：
文献类型：
--
作者：
Zhai, Jixian;Arikit, Siwaret;Meyers, Blake C.
微小RNA（miRNAs）类似于21 nt的小RNA，其与其靶mRNA配对，并且在许多情况下触发切割，特别是在植物中。尽管许多计算工具可以预测miRNA：mRNA相互作用，但由于miRNA在翻译抑制中的功能或由于未经验证的靶预测的高假阳性率（超过90%），验证切割事件仍然至关重要。几年前，三个实验室描述了类似的方法，通过克隆切割或脱帽的mRNA的5'末端来验证miRNA靶的切割。为了利用高通量测序技术的最新进展，我们设计了一种更新的方案，以（1）实现更快的文库制备，以及（2）通过将索引样品汇集在一起进行测序来降低成本。在这里，我们提供了从总RNA开始的帕雷文库构建的分步方案。该方案已在我们的实验室中成功地用于验证各种植物物种中的miRNA靶标。我们还提供了一些常见问题的故障排除建议。(C)2013 Elsevier Inc. All rights reserved.
MicroRNAs (miRNAs) are similar to 21 nt small RNAs that pair to their target mRNAs and in many cases trigger cleavage, particularly in plants. Although many computational tools can predict miRNA:mRNA interactions, it remains critical to validate cleavage events, due to miRNA function in translational repression or due to high rates of false positives (over 90%) for unvalidated target predictions. A few years ago, three laboratories described similar methods to validate cleavage of miRNA targets by the cloning en masse of 5' ends of cleaved or uncapped mRNAs. To take advantage of the recent progress in high-throughput sequencing technology, we have devised an updated protocol to (1) enable much faster library preparation, and (2) reduce the cost by pooling indexed samples together for sequencing. Here we provide a step-by-step protocol for PARE library construction, starting from total RNA. This protocol has been successfully used in our laboratory to validate miRNA targets in a variety of plant species. We also provide advice for troubleshooting on some common issues. (C) 2013 Elsevier Inc. All rights reserved.