A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis

编号 040038003

推送时间 20230130

研究领域 森林培育 

年份 2022 

类型 期刊 

语种 英语

标题 A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis

来源期刊 Plant Methods

期 第380期

发表时间 20221212

关键词 Chromatin-bound RNAs (CB-RNAs);  Transcription regulation;  Co-transcriptional splicing (CTS);  Arabidopsis thaliana; 

摘要 Background
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.
Result
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.
Conclusion
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.

服务人员 孙小满

服务院士 尹伟伦

PDF文件 浏览全文