Multiplexed DNA-FISH imaging dataset from Drosophila embryos at nuclear cycles 11-14. Examples on how to load and use this dataset can be found at this GitHub repository. Data processing details Barcodes were segmented using a neural network (stardist) specifically trained for the detection of 3D diffraction limited spots produced by our microscope. To extract the position of the barcode with sub-pixel accuracy, a subsequent 3D Gaussian fit of the regions segmented by stardist was performed with Big-FISH (https://github.com/fish-quant/big-fish). Barcode localizations with intensities lower than 1.5 times that of the background were filtered out. Nuclei were segmented from projected DAPI images using stardist with a neural network trained for detection of nuclei from Drosophila embryos under our imaging conditions. Barcodes were then attributed to single nuclei by using the XY coordinates of the barcodes and the DAPI masks of the nuclei. Finally, pairwise distance matrices were calculated for each single nucleus.