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

Zhang, Qiqi1,2; Zhao, Fengli2; Wu, Zhe2; Zhu, Danling2

2022-12-12

10.1186/s13007-022-00967-y

PLANT METHODS   Impact Factor & Quartile

1746-4811

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.

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

SCI

English

Corresponding

National Natural Science Foundation of China (NSFC)["JCYJ20190809141201671","20200925153455004","KYTDPT20181011104005"] ; Guangdong Innovation and Entrepreneurial Research Team Program["32270369","31970277","2019KSYS006"] ; Key laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes[32070645] ; [2016ZT06S172]

Biochemistry & Molecular Biology ; Plant Sciences

Biochemical Research Methods ; Plant Sciences

WOS:000895897700001

BMC

Web of Science

1.Harbin Inst Technol, Harbin 150001, Peoples R China
2.Southern Univ Sci & Technol, Guangdong Higher Educ Inst,Sch Life Sci, Inst Plant & Food Res, Key Lab Mol Design Plant Cell Factory,Dept Biol, Shenzhen 518055, Peoples R China

Zhang, Qiqi,Zhao, Fengli,Wu, Zhe,et al. A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis[J]. PLANT METHODS,2022,18(1).

Zhang, Qiqi,Zhao, Fengli,Wu, Zhe,&Zhu, Danling.(2022).A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis.PLANT METHODS,18(1).

Zhang, Qiqi,et al."A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis".PLANT METHODS 18.1(2022).