High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor

We describe a protocol to conduct a high-throughput in vitro processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed by deep sequencing library generation. This comprehensive approach allows the mapping of cleavage sites and the measurement...

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Detalles Bibliográficos
Autores principales: Kim, Kijun, Kim, V. Narry
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8749294/
https://www.ncbi.nlm.nih.gov/pubmed/35036952
http://dx.doi.org/10.1016/j.xpro.2021.101042
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author Kim, Kijun
Kim, V. Narry
author_facet Kim, Kijun
Kim, V. Narry
author_sort Kim, Kijun
collection PubMed
description We describe a protocol to conduct a high-throughput in vitro processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed by deep sequencing library generation. This comprehensive approach allows the mapping of cleavage sites and the measurement of processing efficiency of a large number of substrates simultaneously. Our protocol is readily modifiable to investigate the effects of chemicals and regulatory proteins. Moreover, cis-acting elements can be examined by replacing the wild-type pri-miRNAs with mutant variants. For complete details on the use and execution of this profile, please refer to Kim et al. (2021).
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spelling pubmed-87492942022-01-13 High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor Kim, Kijun Kim, V. Narry STAR Protoc Protocol We describe a protocol to conduct a high-throughput in vitro processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed by deep sequencing library generation. This comprehensive approach allows the mapping of cleavage sites and the measurement of processing efficiency of a large number of substrates simultaneously. Our protocol is readily modifiable to investigate the effects of chemicals and regulatory proteins. Moreover, cis-acting elements can be examined by replacing the wild-type pri-miRNAs with mutant variants. For complete details on the use and execution of this profile, please refer to Kim et al. (2021). Elsevier 2022-01-06 /pmc/articles/PMC8749294/ /pubmed/35036952 http://dx.doi.org/10.1016/j.xpro.2021.101042 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Kim, Kijun
Kim, V. Narry
High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
title High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
title_full High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
title_fullStr High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
title_full_unstemmed High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
title_short High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
title_sort high-throughput in vitro processing of human primary microrna by the recombinant microprocessor
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8749294/
https://www.ncbi.nlm.nih.gov/pubmed/35036952
http://dx.doi.org/10.1016/j.xpro.2021.101042
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