Fig. 1

Strategy of the QeFS experiment. A Barcoded enhancers were cloned into reporter constructs containing degenerate 3′ tags. A sequencing-based approach (see the “Methods” section) associates tags with barcodes (“BC”). P5, P7: Illumina adapters. B A pool of reporter flies was generated and crossed to a marker line enabling FACS isolation of the desired cell types. Targeted RNA- and DNA-sequencing of degenerate tags measured reporter and transcript abundance; DNA-normalized enhancer expression levels were calculated and used for subsequent analysis. C Schematic of enhancer library design. (Left) The 12 endogenous enhancers and relative locations of motifs selected for perturbation are displayed in the left panel. (Middle) An example mutational series is shown for the enhancer CBP2862, including the wildtype, single motif mutants, double mutants and the triple mutant, comprising mutations in the DNA binding motifs of Deaf1, Schlank, and ZIPIC. These enhancers otherwise retain their endogenous sequence context, and other TF binding motifs are maintained in the sequence. (Right) Each enhancer construct is represented by multiple tags in the reporter library, providing internal biological replicates