Luciferase Assay for Insulator Barrier Activity

Insulator barrier activity by luciferase assay was carried out using Bright-Glo™ Luciferase Assay System (Promega)^{16 ,24 (link)}. Individual male larvae were homogenized in 50 μL of Glo Lysis buffer (Promega) and incubated at RT for 10 min. Extracts were cleared from debris by centrifugation. Then, 20 μL of soluble extract was dispensed in a 96-well white flat bottom plate (Costar), and the same volume of Bright-Glo luciferase reagent (Promega) was added to each well. Luciferase signal was quantified using a Spectramax II Gemini EM plate reader (Molecular Devices). Luciferase levels were measured for 12 individual whole third instar male for all genotypes indicated in a single panel simultaneously. Luciferase value was normalized to total protein of each larva determined by BCA reagent (Thermo Scientific). The relative luciferase activity of a population of a single genotype was aggregated into a box and whisker plot. Populations were compared with one-way ANOVA followed by a Tukey HSD post hoc test to obtain P values for each pairwise comparison. The P values for pairwise comparisons between the control and RNAi lines within both non-insulated and insulated groups are listed in Supplementary Table 4. All values of relative luciferase activity are available in Source Data 7.