Target-enrichment sequencing for detailed characterization of small RNAs.
Nat Protoc
; 13(4): 768-786, 2018 04.
Article
en En
| MEDLINE
| ID: mdl-29565901
ABSTRACT
Identification of important, functional small RNA (sRNA) species is currently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method, termed target-enrichment of sRNAs (TEsR), that enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full-length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNAs, removal of nontargeted sRNAs and sequencing. By this approach, target sRNAs can be enriched by a factor of 500-30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5' cap or of secondary structures or abundance levels. Moreover, TEsR allows the detection of the complete sequence (including sequence variants, and 5' and 3' ends) of precursors, as well as intermediate and mature forms, in a quantitative manner. A well-trained molecular biologist can complete the TEsR procedure, from RNA extraction to sequencing library preparation, within 4-6 d.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Análisis de Secuencia de ARN
/
Técnicas de Amplificación de Ácido Nucleico
/
ARN Pequeño no Traducido
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Nat Protoc
Año:
2018
Tipo del documento:
Article
País de afiliación:
Japón