Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue [plasmid]

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.