GSM6692786: ATAC hda6/ldl1/2 rep1; Arabidopsis thaliana; ATAC-seq
2 g of 14-day-old Arabidopsis seedlings were collected immediately before the nuclear extraction procedure. Seedlings were sequentially chopped using a sharp razor blade with 5 ml cold NEB buffer on ice. The slurry was filtered through 4 layers of Miracloth and a 20-μm CellTrics twice to remove large debris, followed by centrifugation at 800g for 10 min at 4°C. The supernatant was removed without disturbing the green pellet and gently resuspended by 200 μl ORB (with 1% Triton X-100). The nuclei suspension was filtered through a 10-μm CellTrics twice to remove aggregate nuclei and debris and transferred to the top of the Sucrose-Percoll gradient (400 μl 60% Percoll in ORB and 200 μl SCB buffer), followed by centrifuging at 1,000g for 15 min at 4°C for nuclei enrichment. The nuclei located at the Sucrose-Percoll interface were collected. Some nuclei were taken and stained with DAPI (0.3 μg/ml) to examine the integrity under microscopy using excitation wavelength at 353 nm and emission wavelength at 465 nm for DAPI. The nuclei number was estimated by a hemocytometer. The Tn5 transposition was carried out by Nextera DNA Library Preparation Kit (Illumina) according to the manufacturer's manual. The 5,000 nuclei were suspended in the transposition reaction mix and incubated at 37°C for 30 min in an Eppendorf Thermo Mixer Comfort (Eppendorf) with agitation at 1,000 rpm for 2 min and resting for 2 min. The transposed DNA fragments were purified immediately after with a MinElute PCR Purification Kit (Qiagen). The purified transposed DNA fragments were amplified by Nextera Index Kit (Illumina) and NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs) for the first PCR amplification with 5 cycles. The KAPA Library Quantification Kit is used for the quantitative PCR to estimate the secondary PCR amplification. The libraries were purified with AMPure XP beads (Beckman Coulter)