NAD tagSeq for transcriptome-wide identification and characterization of NAD+-capped RNAs
Autor: | Huan Zhong, Zhu Yang, Yiji Xia, Xiaojian Shao, Zongwei Cai, Hailei Zhang |
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Rok vydání: | 2020 |
Předmět: |
0303 health sciences
RNA Stability Chemistry Sequence analysis RNA Computational biology General Biochemistry Genetics and Molecular Biology 03 medical and health sciences 0302 clinical medicine Transcription (biology) Complementary DNA Nanopore sequencing NAD+ kinase RNA extraction 030217 neurology & neurosurgery 030304 developmental biology |
Zdroj: | Nature Protocols. 15:2813-2836 |
ISSN: | 1750-2799 1754-2189 |
Popis: | 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. NAD+ is one of the noncanonical nucleotides recently found to cap the 5′ end of RNAs. This Protocol Extension describes procedures for genome-wide analysis of NAD+-capped RNAs by direct RNA sequencing on an Oxford Nanopore platform. |
Databáze: | OpenAIRE |
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