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

Xiaojian Shao, Hailei Zhang, Zhu Yang, Huan Zhong, Yiji Xia*, Zongwei Cai*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

13 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)2813-2836
Number of pages24
JournalNature Protocols
Issue number9
Early online date3 Aug 2020
Publication statusPublished - Sept 2020

Scopus Subject Areas

  • Biochemistry, Genetics and Molecular Biology(all)

User-Defined Keywords

  • Gene Expression Profiling
  • NAD
  • RNA Caps
  • Sequence Analysis, RNA
  • Staining and Labeling


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