High-throughput functional analysis of regulatory variants using a massively parallel reporter assay

• Published in: STAR protocols 2023-12, Vol.4 (4), p.102731-102731, Article 102731
• Main Authors: Delfosse, Kate, Gerhardinger, Chiara, Rinn, John L., Maass, Philipp G.
• Format: Article
• Language: English
• Subjects: Gene Expression; Genetics; Genomics; High Throughput Screening; Plasmids - genetics; Protocol; Systems biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102731

Association studies describe genetic associations between noncoding variants and disease susceptibility; however, they do not provide functional insight into the underlying molecular mechanisms of these variants. We present a protocol to assay the regulatory potential of thousands of noncoding variants using massively parallel reporter assays. We describe steps for oligo design, generating a plasmid pool, and extracting tag-seq libraries from cells to quantify the tested sequences. For complete details on the use and execution of this protocol, please refer to Oliveros and Delfosse et al.1 [Display omitted] •Key steps for performing massively parallel reporter assays (MPRAs)•Protocol for high-throughput cloning, transfection, and MPRA library preparation•MPRA design to assay thousands of regulatory regions and/or genetic variants Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Association studies describe genetic associations between noncoding variants and disease susceptibility; however, they do not provide functional insight into the underlying molecular mechanisms of these variants. We present a protocol to assay the regulatory potential of thousands of noncoding variants using massively parallel reporter assays. We describe steps for oligo design, generating a plasmid pool, and extracting tag-seq libraries from cells to quantify the tested sequences.