GSM8807667: Tak-1_ABA_ATAC, rep2; Marchantia polymorpha; ATAC-seq
Whole Marchantia gemmalings (1.5 g) were finely chopped with a razor blade in 4 mL of ice-cold Galbraith buffer (45 mM MgCl₂, 20 mM MOPS, 30 mM sodium citrate, 0.2% Triton X-100, and cOmplete Protease Inhibitor Cocktail) for 2-5 minutes in a chilled petri dish, followed by 10 minutes of incubation at 4°C. The lysate was then filtered twice through a 40 µm filter and transferred to an ice-cold 15 mL tube. Nuclei were pelleted by centrifugation at 500 × g for 10 minutes at 4°C, and the supernatant was carefully removed. The pellet was gently resuspended in 2 mL of Galbraith buffer and centrifuged again under the same condition. After removing the supernatant, the nuclei were resuspended in 1 mL of Galbraith buffer in a 5 mL round-bottom tube and stained with 10 µg/mL DAPI. Flow cytometry was performed on a BD FACS Aria II using a 100 µm nozzle at a pressure below 20 psi. Sorting gates were set based on FSC and SSC to exclude debris and potential doublets. Baseline fluorescence was established by running unstained control samples before sorting DAPI-stained nuclei. 50,000 sorted nuclei were collected into 500 µL of Galbraith buffer (without Triton X-100) in 1.5 mL Eppendorf tubes per sample. The nuclei were pelleted by centrifugation at 1000 × g for 10 minutes at 4°C. The sorted nuclei were resuspended in a 50 µL tagmentation reaction mixture consisting of 25 µL of 2x TD buffer, 2.5 µL of TDE1 enzyme (Illumina Tagment DNA TDE1 Enzyme and Buffer Kits), and 22.5 µL of nuclease-free water. The reaction was incubated at 37°C for 30 minutes, followed by DNA purification with Zymo CHIP DNA Clean and Concentrator (Cat# D5205). The eluted DNA was amplified with dual-indexed Nextera primers using NEBNext 2x HiFi PCR Master Mix (New England Biolabs) for library construction. For library amplification, 10 µL of transposed DNA fragments were mixed with 25 µL of NEBNext 2x HiFi PCR Master Mix, 6.25 µL of 10 µM T5 primer, and 2.5 µL of nuclease-free water. The corresponding T7 primer (6.25 µL) was added, and PCR amplification was performed with the following conditions: 72°C for 5 minutes (initial denaturation), 98°C for 30 seconds, then 5 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. Additional cycles were optimized via qPCR using PowerSYBR™ Green Master Mix on 5 µL of the amplified library. Amplified libraries underwent dual size selection using a 1.2x SPRIselect magnetic bead ratio (Beckman Coulter). Library quality was assessed with a Bioanalyzer High Sensitivity DNA Analysis kit (Agilent).