lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements

• Published in: Nature protocols Vol. 15; no. 8; pp. 2387 - 2412
• Main Authors: Gordon, M. Grace, Inoue, Fumitaka, Martin, Beth, Schubach, Max, Agarwal, Vikram, Whalen, Sean, Feng, Shiyun, Zhao, Jingjing, Ashuach, Tal, Ziffra, Ryan, Kreimer, Anat, Georgakopoulos-Soares, Ilias, Yosef, Nir, Ye, Chun Jimmie, Pollard, Katherine S., Shendure, Jay, Kircher, Martin, Ahituv, Nadav
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-08-2020; Nature Publishing Group
• Subjects: 631/1647/2017; 631/1647/514/2254; 631/1647/794; 631/208/200; 631/61/191; Analytical Chemistry; Bar codes; Base Sequence; Biological Techniques; Biomedical and Life Sciences; Computational Biology/Bioinformatics; Computer applications; Data processing; Deoxyribonucleic acid; DNA; DNA sequencing; Genetic regulation; Genomes; Health sciences; Lentivirus - genetics; Life Sciences; Methods; Microarrays; Nucleotide sequence; Nucleotide sequencing; Organic Chemistry; Physiological aspects; Protocol; Regulatory sequences; Regulatory Sequences, Nucleic Acid - genetics; Reporter gene; Ribonucleic acid; RNA; Sequence Analysis, DNA - methods; Switches; Transcription; Workflow; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-020-0333-5

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.
This protocol describes a lentivirus-based massively parallel reporter assay (lentiMPRA) for quantitative assessment of the activity of gene regulatory elements and an accompanying Nextflow-based computational pipeline for analysis.