Embryos were collected and washed prior to mounting in halocarbon oil on a glass bottom dish. Real time staging of embryos was possible through the His2av-RFP marker used to measure nuclear density (nuclei/2500µm2). Live imaging was performed on a Nikon Eclipse Ti2 inverted fluorescent microscope. Embryos were processed for ATACseq exactly 15 min into NC14. For optogenetic experiments, His2Av-RFP and CBPCRY2;His2Av-RFP embryos were treated with a repeating sequence of 60s exposure to 470nm filtered light followed immediately by a 10 ms exposure to 555nm. This sequence was initiated upon entry into NC10 and ended 15 min into nuclear-cycle 14. Staged single embryos were picked and homogenized in 10µL of pre-chilled lysis buffer (10mM Tris pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1%NP-40). Embryo homogenate was resuspended by adding an additional 40µL of lysis buffer and spun down at 500g for 10 min at 4˚C. The supernatant was removed under a dissecting scope to ensure the small pellet was not disturbed. 2.5µL of water, 2.5µL of Tn5 enzyme, and 5µL of TD buffer (Illumina Tagment DNA Enzyme and Buffer Kit) were added to resuspend the pellet, followed by a 37˚C incubation for 30 min. ATAC-seq in S2 cells was carried out similarly, in which 200,000 cells were spun down for 3min at 600g at 4˚C, washed with 1X PBS, and resuspended in 100μL lysis buffer. Lysed cell material was pelleted after centrifuging for 10min at 600g at 4˚C. The supernatant was replaced with 47.5UL TD buffer and 2.5 Tn5 enzyme (Illumina) and incubated at 37˚C for exactly 30min. Libraries were prepared using IDT for Illumina Nextera DNA Unique Dual indices, and PCR amplified with NEBNext Hi-Fi 2x PCR Master Mix (72˚C for 5min, 98˚C for 30s, followed by 12 cycles of 98˚C for 10s, 63˚C for 30s, and 72˚C for 1min). Libraries were DNA purified using magnetic axygen beads and washed with ethanol (Thermo Fisher Scientific).