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Global in situ profiling of RNA-RNA spatial interactions with RIC-seq

Abstract

Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix all the protein-mediated RNA-RNA interactions in situ. After cell permeabilization and micrococcal nuclease digestion, the proximally interacting RNAs are 3′ end-labeled with pCp-biotin and subsequently ligated using T4 RNA ligase. The chimeric RNAs are then enriched and converted into libraries for paired-end sequencing. After deep sequencing, computational analysis yields interaction strength scores for every base on proximally interacting RNAs in the starting populations. The whole experimental procedure is designed to be completed within 6 d, followed by an additional 8 d for computational analysis. RIC-seq technology can unbiasedly detect intra- and intermolecular RNA-RNA interactions, thereby rendering it useful for reconstructing RNA higher-order structures and identifying direct noncoding RNA targets.

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Fig. 1: Overview of RIC-seq technology.
Fig. 2: Evaluation of RIC-seq library.
Fig. 3: Inter- and intramolecular RNA-RNA interactions revealed by RIC-seq.

Data availability

RIC-seq data for HeLa cells (originally published in ref. 36) are available in the Gene Expression Omnibus under accession number GSE127188. An example dataset is deposited at https://drive.google.com/drive/folders/1HFjcE2LwbPFsmQy4h39wBZb2 cKCJIyB-?usp=sharing.

Code availability

The scripts for RIC-seq data analysis can be freely downloaded from GitHub at https://github.com/caochch/RICpipe.

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Acknowledgements

We thank Jing Hu for the critical review of this manuscript. This work was supported by the Ministry of Science and Technology of China (2017YFA0504400), the National Natural Science Foundation of China (32025008, 91740201, 91940306, 31522015 and 81921003), the Strategic Priority Program of CAS (XDB37000000; to Y.X.), the Beijing Municipal Natural Science Foundation (5182024) and the National Natural Science Foundation of China (31900465; to C.C).

Author information

Affiliations

Authors

Contributions

Z.C. and Y.X. designed, implemented and optimized the original RIC-seq experimental protocol with the help of R.Y. and R.S. C.C. designed the data-processing pipeline. N.H. and H.Z. tested the computational pipeline. Y.X., C.C. and Z.C. prepared the manuscript.

Corresponding author

Correspondence to Yuanchao Xue.

Ethics declarations

Competing interests

C.C., Z.C. and Y.X. have filed a patent application for RIC-seq technology with the application number 201910384194.2.

Additional information

Peer review information Nature Protocols thanks Rory Johnson, John Rinn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key reference using this protocol

Cai, Z. et al. Nature 582, 432–437 (2020): https://doi.org/10.1038/s41586-020-2249-1

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Cao, C., Cai, Z., Ye, R. et al. Global in situ profiling of RNA-RNA spatial interactions with RIC-seq. Nat Protoc 16, 2916–2946 (2021). https://doi.org/10.1038/s41596-021-00524-2

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