title: Massively parallel reporter assays in cultured mammalian cells.
abstract: The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.
authors.affiliation: Broad Institute.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Melnikov
authors.affiliation: Broad Institute.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Broad Institute.
authors.forename: Peter
authors.initials: P
authors.lastname: Rogov
authors.affiliation: Broad Institute.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Broad Institute; tarjei@broadinstitute.org.
authors.forename: Tarjei S
authors.initials: TS
authors.lastname: Mikkelsen
mesh_terms: D018929:Cell Culture Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D015345:Oligonucleotide Probes