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

ABSTRACT: 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).

SUBMITTER: Kim K

PROVIDER: S-EPMC8749294 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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

Kim Kijun K Kim V Narry VN

STAR protocols 20220106 1

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</i ...[more]

PMID: 35036952

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