BioProject

Activity of enhancers in Drosophila embryos was measured by highly parallel reporter assay. More...

Activity of enhancers in Drosophila embryos was measured by highly parallel reporter assay. We examined the results of mutating binding sites for 4 poorly studied TFs individually or in combination, and characterized complex genetic interactions among the different classes of motif mutant. Overall design: We generated a population of Drosophila embryos in each of which reporter gene expression is driven by one genomically integrated reporter element from a pool of wild type enhancers or enhancers in which all binding sites for one or more selected TFs have been mutated. Each tested enhancer was tagged with several distinct 20-base degenerate sequence tags in the 3' UTR of the reporter gene. We dissociated embryonic cells and FACS-sorted informative populations, and reporter gene molecules were counted by a Unique Molecular Identifier (UMI) amplification strategy. We detected RNA and DNA levels by high-throughput sequencing of tagged reporter gene amplicons and normalized gene expression (RNA UMI counts) by dividing by DNA UMI counts to generate a measure of enhancer activity. Less...