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  • Protocol
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Full-length circular RNA profiling by nanopore sequencing with CIRI-long


Circular RNAs (circRNAs) have important roles in regulating developmental processes and disease progression. As most circRNA sequences are highly similar to their cognate linear transcripts, the current short-read sequencing-based methods rely on the back-spliced junction signal for distinguishing circular and linear reads, which does not allow circRNAs’ full-length structure to be effectively reconstructed. Here we describe a long-read sequencing-based protocol, CIRI-long, for the detection of full-length circular RNAs. The CIRI-long protocol combines rolling circular reverse transcription and nanopore sequencing to capture full-length circRNA sequences. After poly(A) tailing, RNase R treatment, and size selection of polymerase chain reaction products, CIRI-long achieves an increased percentage (6%) of circular reads in the constructed library, which is 20-fold higher compared with previous Illumina-based strategies. This method can be applied in cell lines or tissue samples, enabling accurate detection of full-length circRNAs in the range of 100–3,000 bp. The entire protocol can be completed in 1 d, and can be scaled up for large-scale analysis using the nanopore barcoding kit and PromethION sequencing device. CIRI-long can serve as an effective and user-friendly protocol for characterizing full-length circRNAs, generating direct and convincing evidence for the existence of detected circRNAs. The analytical pipeline offers convenient functions for identification of full-length circRNA isoforms and integration of multiple datasets. The assembled full-length transcripts and their splicing patterns provide indispensable information to explore the biological function of circRNAs.

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Fig. 1: Overview of the CIRI-long protocol.
Fig. 2: Applications of the CIRI-long protocol.
Fig. 3: RNA hydrolysis and TS during circular RT.
Fig. 4: Overview of the CIRI-long data analysis workflow.
Fig. 5: Anticipated library size after beads selection.

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Data availability

The CIRI-long dataset used in this manuscript can be downloaded from the National Genomics Data Center46 (China National Center for Bioinformation: with accession number CRR194209.

Code availability

The CIRI-long software can be downloaded from GitHub at


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This work was supported by grants from the National Natural Science Foundation of China (32130020, 32025009, 91940306, 32200530) and the National Key R&D Project (2021YFA1300500, 2021YFA1302000).

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Authors and Affiliations



L.H. and J.Z. developed the experimental protocol for CIRI-long. L.H., J.Z. and F.Z. wrote the manuscript. F.Z. conceived the study.

Corresponding authors

Correspondence to Jinyang Zhang or Fangqing Zhao.

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Nature Protocols thanks Michael Clark and Leng Han for their contribution to the peer review of this work.

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Zhang, J. et al. Nat. Biotechnol. 39, 836–845 (2021):

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Hou, L., Zhang, J. & Zhao, F. Full-length circular RNA profiling by nanopore sequencing with CIRI-long. Nat Protoc 18, 1795–1813 (2023).

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