Abstract
The Microprocessor complex is crucial in microRNA (miRNA) biogenesis, as it processes primary miRNAs (pri-miRNAs) into precursor miRNAs. Here, we present a high-throughput, radioisotope-free protocol for studying pri-miRNA processing using randomized sequences. We describe steps for randomized substrate preparation, protein purification, processing assays, and DNA library construction for sequencing. This technique explores pri-miRNA processing, uncovers key RNA elements, and illuminates gene expression regulation. However, its efficacy may be constrained by data analysis complexity and the requirement for specialized equipment. For complete details on the use and execution of this protocol, please refer to Nguyen et al. (2023).1.