A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis.
A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis.
复制标题
一种分离和分析拟南芥染色质结合RNA的简单而可靠的方法。
DOI：
10.1186/s13007-022-00967-y
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发表时间：
2022-12-12
期刊：
影响因子：
5.1
通讯作者：
Zhu, Danling
中科院分区：
文献类型：
--
作者：
Zhang, Qiqi;Zhao, Fengli;Wu, Zhe;Zhu, Danling
关键词：
染色质结合的RNA是转录的主要产物，经过染色质上的处理，如封端、剪接和多聚腺苷化。然后这些处理步骤决定了RNA的命运。尽管它至关重要，但在植物中还缺乏一种简单而可靠的方法来分离染色质结合的不同部分的RNA。在这里，我们描述了我们的更新方法和相关的逐步协议，以分离染色质结合的RNA在A.thaliana。染色质结合RNA的分离是基于1M尿素洗涤，该洗涤将大多数非染色质相关蛋白从细胞核中移除，这是以前在哺乳动物细胞中开发的。根据需要，分离的染色质结合的RNA既可以直接用于基因特异性分析，也可以进行进一步的rRNA去除，也可以选择多腺化RNA去除，然后进行高通量测序。还提供了这些程序的详细协议。对去掉和没有去掉多聚腺苷化RNA的染色质结合RNA的测序结果进行了比较，发现一小部分CB-RNAs是多腺化的，但还没有完全剪接，代表了RNA加工的中间染色质。这种优化的染色质结合RNA纯化方法简单、可靠，可用于植物转录及其偶联RNA加工的研究。网上版载有补充材料，可在10.1186/s13007-022-00967-y查阅。
Chromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fractions of chromatin-bound RNAs is missing in plants. Here, we describe our updated method and the associated step-by-step protocol for chromatin-bound RNAs isolation in A. thaliana. The chromatin-bound RNAs isolation is based on the 1 M UREA wash that removes the majority of non-chromatin-associated proteins from the nucleus, as previously developed in mammalian cells. On-demand, the isolated chromatin-bound RNAs can be either used directly for gene-specific analysis or subject to further rRNA removal and also the optional polyadenylated RNA removal, followed by high-throughput sequencing. Detailed protocols for these procedures are also provided. Comparison of sequencing results of chromatin-bound RNAs with and without polyadenylated RNA removal revealed that a small fraction of CB-RNAs is polyadenylated but not yet fully spliced, representing RNA-processing intermediate on-chromatin. This optimized chromatin-bound RNAs purification method is simple and robust and can be used to study transcription and its-coupled RNA processing in plants. The online version contains supplementary material available at 10.1186/s13007-022-00967-y.
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