Abstract：
: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.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Shao X,Zhang H,Yang Z,Zhong H,Xia Y,Cai Zdoi
10.1038/s41596-020-0363-zsubject
Has Abstractpub_date
2020-09-01 00:00:00pages
2813-2836issue
9eissn
1754-2189issn
1750-2799pii
10.1038/s41596-020-0363-zjournal_volume
15pub_type
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