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Revealing nascent RNA processing dynamics with nano-COP


During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3′-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3′ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.

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Fig. 1: nano-COP schematic.
Fig. 2: Direct RNA sequencing of 4sU-labeled chromatin-associated RNA provides the most accurate measurement of the nascent transcriptome.
Fig. 3: Troubleshooting the incubation time for 3′ end poly(A) tailing with oGAB11.
Fig. 4: Detection of poly(A) and poly(I) tails in ONT direct RNA sequencing data.
Fig. 5: Representative RT-qPCR plots of RNA purified by cellular fractionation with varying incubation times in the presence of the splicing inhibitor pladienolide B (PlaB).
Fig. 6: Nano-COP captures the nascent transcriptome.

Data availability

The accession numbers for the nanopore sequencing data presented in this paper are Gene Expression Omnibus (GEO): GSE123191 (data from ref. 18) and GSE154079. Supplementary Table 1 indicates the correct accession number for each sample. Source data are provided with this paper.

Code availability

All scripts for data analyses described in this paper are available at The code for nanopolish-detect-polyI is available at


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We thank members of the Churchman lab, F. Winston, W. Timp, R. Workman, N. Sadowski, M. Marin, B. Smalec, M. Richardson, R. Ietswaart, and A. Markham for helpful discussions, advice, and assistance and C. Kaplan, J. Bridgers, B. Smalec, J. Falk and C. Patil for critical reading of the manuscript. This work was supported by the NIH (R21-HG009264, R01-HG010538, and R01-GM117333 to L.S.C.; F31-GM122133 to H.L.D.), an NSF Graduate Research Fellowship to H.E.M., the Fonds de Recherche du Québec–Santé and the Canadian Institutes of Health Research (Post-doctoral fellowship awards to K.C.). J.T.S. is supported by the Ontario Institute for Cancer Research through funds provided by the Government of Ontario and the Government of Canada through Genome Canada and Ontario Genomics (OGI-136).

Author information




H.L.D., K.C. and L.S.C. conceived and designed the study. H.L.D. established the nano-COP protocol and K.C. developed the poly(I) tailing approach. H.L.D., K.C. and H.E.M. performed experiments. H.L.D. and K.C. generated scripts and performed data analysis. P.S.T. and J.T.S. developed nanopolish-detect-polyI. H.L.D., K.C., H.E.M. and L.S.C. wrote the manuscript. P.S.T. and J.T.S. reviewed and edited the manuscript.

Corresponding author

Correspondence to L. Stirling Churchman.

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Competing interests

J.T.S. receives research funding from Oxford Nanopore Technologies. J.T.S. and H.L.D. have received travel support to attend and speak at meetings organized by Oxford Nanopore Technologies. All other authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Shobbir Hussain, Jixian Zhai 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.

Related links

Key reference using this protocol:

Drexler, H. L., Choquet, K. & Churchman, L. S. Mol. Cell 77, 985–998.e8 (2020):

This protocol is an extension to: Nat. Protoc. 11, 813–833 (2016):

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Fig. 1, Supplementary Note and Supplementary Table 1

Source data

Source Data Fig. 3

Unprocessed gel.

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Drexler, H.L., Choquet, K., Merens, H.E. et al. Revealing nascent RNA processing dynamics with nano-COP. Nat Protoc 16, 1343–1375 (2021).

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