Dragonfly spinning disc microscope

The Oligopaint FISH libraries were chosen from the database (Probes for Human genome, build 38. Mining Settings: ‘Balance’) generated by the Wu Lab (https://oligopaints.hms.harvard.edu/). Probe sets were designed using standard procedures to target the ecDNA localized genes defined in Supporting Information Table S10. For the primary oligo pool, we purchased the Oligoarray pool (Synbio Technologies), and prepared FISH probes as described previously.⁸² The Oligopaint FISH staining procedure was carried out following the previous description.⁸³ Images of nuclei were collected with a Dragonfly spinning disc microscope (Andor), processed by Fiji (Fiji Is Just ImageJ, software available at https://imagej.net/Fiji/Downloads), and analysed by Imaris version 8.4.1 (Bitplane Inc., software available at http://www.bitplane.com/).

Embryos were mounted in 35 mm glass bottom dishes using 0.7% low melting point agarose. Confocal z-stacks were acquired on a LSM 710 Meta BiG (Zeiss) and a LSM 880 FastAiry (Zeiss) using a LD C-Apochromat W/40×1.1 NA objective. Timelapse imaging was performed on a Dragonfly Spinning Disc microscope (Andor) using Apo λ LWD W/40×1.15 NA. Bright-field images of the whole-mount zebrafish embryos were taken on a Nikon SMZ1270i with Tucsen Michrome 6 camera. Movies of blood flow (Movies 4-7) were recorded with a Prime BSI Express camera at 100 frames per second and captured using a EC-Plan Neofluar 20×0.8 NA objective on an Axio Observer 7 inverted microscope (Zeiss).