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FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

Nature Protocols volume 16, pages 4355–4381 (2021)Cite this article

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Abstract

Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform.

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Fig. 1: Overview of FLEP-seq protocol.
Fig. 2: Bioinformatics workflow.
Fig. 3: Application of FLEP-seq.
Fig. 4: An example of subcellular protein fractionation from 12-d-old Arabidopsis seedlings.
Fig. 5: The RNA fractions extracted from 12-d-old Arabidopsis seedlings.
Fig. 6: cDNA amplification product after rRNA depletion.
Fig. 7: Examples of bioanalyzer plots.
Fig. 8: Nanopore sequencing run statistics for reads yield and read length.
Fig. 9: The performance of PolyAcaller on Nanopore data.

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Data availability

The data used in this study were previously published35 and deposited at NCBI under the accession number PRJNA591665.

Code availability

All software used in this study is described in detail in the Equipment section. Custom Python and R code used for bioinformatics analysis have been deposited in a GitHub repository (https://github.com/ZhaiLab-SUSTech/FLEPSeq).

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Acknowledgements

We are grateful for the useful comments and edits suggested by the anonymous reviewers. The group of J.Z. is supported by a Stable Support Plan Program of Shenzhen Natural Science Fund Grant (20200925153345004), a National Key R&D Program of China Grant (2019YFA0903903), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S172), the Shenzhen Sci-Tech Fund (KYTDPT20181011104005) and the Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes (2019KSYS006).

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Author notes

  1. These authors contributed equally: Yanping Long, Jinbu Jia.

Authors and Affiliations

  1. Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Institute of Plant and Food Science, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Yanping Long, Jinbu Jia, Weipeng Mo, Xianhao Jin & Jixian Zhai

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  1. Yanping Long
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Contributions

Y.L., J.J. and J.Z. conceived and designed the study. Y.L., J.J. and X.J. developed the method and performed the experiments. J.J., W.M. and J.Z. designed the computational pipeline and analyzed the data. J.Z. conceived and oversaw the study. All authors wrote and revised the manuscript.

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Correspondence to Jixian Zhai.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Zongwei Cai, Shona Murphy, Yongsheng Shi and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Jia et al. Nat. Plants 6, 780–788 (2020): https://doi.org/10.1038/s41477-020-0688-1

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Jia et al. Nat. Plants 6, 780–788 (2020): https://doi.org/10.1038/s41477-020-0688-1

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Long, Y., Jia, J., Mo, W. et al. FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing. Nat Protoc 16, 4355–4381 (2021). https://doi.org/10.1038/s41596-021-00581-7

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