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NAD tagSeq for transcriptome-wide identification and characterization of NAD+-capped RNAs


Several noncanonical initial nucleotides (NCINs) have been found to cap RNAs and possibly regulate RNA stability, transcription and translation. NAD+ is one of the NCINs that has recently been discovered to cap RNAs in a wide range of species. Identification of the NAD+-capped RNAs (NAD-RNAs) could help to unveil the cap-mediated regulation mechanisms. We previously reported a method termed NAD tagSeq for genome-wide analysis of NAD-RNAs. NAD tagSeq is based on the previously published NAD captureSeq protocol, which applies an enzymatic reaction and a click chemistry reaction to label NAD-RNAs with biotin followed by enrichment with streptavidin resin and identification by RNA sequencing. In the current NAD tagSeq method, NAD-RNAs are labeled with a synthetic RNA tag and identified by direct RNA sequencing based on Oxford Nanopore technology. Compared to NAD captureSeq, NAD tagSeq provides a simpler procedure for direct sequencing of NAD-RNAs and noncapped RNAs and affords information on the whole sequence organization of NAD-RNAs and the ratio of NAD-RNAs to total transcripts. Furthermore, NAD-RNAs can be enriched by hybridizing a complementary DNA probe to the RNA tag, thus increasing the sequencing coverage of NAD-RNAs. The strategy of tagging RNAs with a synthetic RNA tag and identifying them by direct RNA sequencing might be employed in analyzing other NCIN-capped RNAs. The experimental procedure of NAD tagSeq, including RNA extraction, RNA tagging and direct RNA sequencing, takes ~5 d, and initial data analysis can be completed within 2 d.

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Fig. 1: Schema of NAD tagSeq.
Fig. 2: Gel electrophoresis of the purified model NAD-RNA.
Fig. 3: Gel analysis of ligation of model NAD-RNA with tagRNA-azide to obtain tagRNA-NAD-RNA using 10% (wt/vol) denaturing PAGE gel.
Fig. 4: RNA integrity analysis using agarose gel electrophoresis, showing 28S rRNA, 18S rRNA and 5S rRNA from A. thaliana seedling.
Fig. 5: IGV screenshot showing a gene and its corresponding NAD-RNA and noncapped-RNA reads.

Data availability

The data sets analyzed with the current protocol are available in the Gene Expression Omnibus repository under the accession number GSE127755. The web links for the raw data are, and

Code availability

All the software used in this protocol, including our Python script for sorting tagged and untagged RNA, are available in the ‘TagSeqTools’ repository under the Apache License v2.0 ( and as Supplementary Software.


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This work was supported by the National Natural Science Foundation of China (91543202 and 21705137 to Z.C.), Hong Kong Baptist University (SDF 15-1012-P04 and RC-ICRS/16-17/04 to Y.X. and Z.C.), the National Key R&D Program of China (2017YFC1600500 to Z.C.) and the Research Grants Council of Hong Kong (GRF grant nos. 12100415, 12100018, 12100717, 12102719, C2009-19GF and AoE/M-403/16 to Y.X.).

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Authors and Affiliations



All authors contributed to the design of the experiments; H. Zhang and X.S. performed experiments; H. Zhong and Z.Y. performed data analysis; all authors analyzed experimental results and contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Yiji Xia or Zongwei Cai.

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The authors declare no competing interests.

Additional information

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

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Key reference using this protocol

Zhang, H. et al. Proc. Natl Acad. Sci. USA 116, 12072–12077 (2019):

This protocol is an extension to Nat. Protoc. 12, 122–149 (2017):

Supplementary information

Supplementary Information

Supplementary Methods and Supplementary Fig. 1.

Reporting Summary

Supplementary Software

Software for processing NAD tagSeq data and demo files.

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Shao, X., Zhang, H., Yang, Z. et al. NAD tagSeq for transcriptome-wide identification and characterization of NAD+-capped RNAs. Nat Protoc 15, 2813–2836 (2020).

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