Work

Protocol for assessing regulatory elements in murine heart using an AAV9-based massively parallel reporter assay

Year: 2025

Type: article

Abstract: The mammalian genome contains thousands of regulatory elements whose activities are highly sensitive to external and internal stimulation. Here, we present a protocol to assess the murine cardiac function of regulatory elements using an adeno-associated virus serotype 9 (AAV9)-based massively parallel reporter assay (MPRA). We describe steps for the selection principle of enhancer candidates, MPRA library construction, animal delivery, sequencing library generations, and data analysis pipeline. Our MPRA protocol provides a comprehensive workflow for enhancer activity quantification in vivo . For complete details on the use and execution of this protocol, please refer to Yangpo Cao et al. 1 • Guidance on self-priming approach to extend the coverage of enhancer regions • Steps for adding UMI to remove the PCR duplicates • Instructions for injecting AAV carrying the library into neonatal mice • Instructions for user-friendly informatics platform for data analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The mammalian genome contains thousands of regulatory elements whose activities are highly sensitive to external and internal stimulation. Here, we present a protocol to assess the murine cardiac function of regulatory elements using an adeno-associated virus serotype 9 (AAV9)-based massively parallel reporter assay (MPRA). We describe steps for the selection principle of enhancer candidates, MPRA library construction, animal delivery, sequencing library generations, and data analysis pipeline. Our MPRA protocol provides a comprehensive workflow for enhancer activity quantification in vivo .

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.103782