DM1-DM2d-Shoot-ATAC-12DAS_1
12 DAS DM1-DM2d and Col-0, 14 DAS DM6-DM7 and Col-0 shoots were used for ATAC-Seq. Nuclei were isolated from 0.05g LN snap-frozen plant tissues utilizing methods from Giuliano et al., 1988 and Dorrity et al., 2021. Tissues were pulverized at 30Hz for 40s with three 3mm beads in a TissueLyser II (Qiagen) within a 2mL EP tube, with subsequent procedures conducted on ice and centrifugation at 4C. Buffer A (1.5mL; 0.8 M sucrose, 10 mM MgCl2, 25 mM Tris-HCl pH 8.0, 1x Protease Inhibitor) was quickly added to the powdered tissue, then gently mixed. After filtration through 40m and 10m strainers, the mixture was centrifuged at 2000  g for 5 minutes. The resulting pellet was resuspended in 1mL Buffer B (0.4 M sucrose, 10 mM MgCl2, 25 mM Tris-HCl pH 8.0, 1x Protease Inhibitor, 1% Triton X-100), then loaded over a 25/75 Percoll gradient. Following a 2500  g, 15 min centrifugation, nuclei were harvested from the 25/75 interface. This nuclear material was re-loaded over 25% Percoll and centrifuged again at 2500  g for 5 minutes. The supernatant was discarded, and the pellet was washed with 1mL Buffer B, typically yielding 100k nuclei per 0.1g tissue. For ATAC-Seq library preparation, each biological sample utilized 50k nuclei. Libraries were prepared using the Hyperactive ATAC-Seq Library Prep Kit (Vazyme TD711-01) and sequenced by Azenta or Novogene.