Abstract
Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
