GSM4681512: 24 hour rep 1 [ATAC-Seq]; Medicago truncatula; ATAC-seq
Before nuclei isolation, all materials were precooled to 4°C. Briefly, roots were chopped for 2 min in 1 ml of pre-chilled lysis buffer (15 mM Tris-HCl pH7.5, 2mM EDTA, 20 mM NaCl, 80 mM KCl, 0.5 mM spermine, 15 mM 2-ME, 0.15 % TritonX-100) in a cold room. This step was repeated four times with a 1 min interval. The homogenate was filtered through one layer of pre-wetted Miracloth, loaded on the surface of a 2 mL dense sucrose buffer (1.7 M sucrose, 10 mM Tris-HCl pH8.0, 2 mM MgCl2, 5 mM 2-ME, 1 mM EDTA, 0.15 % Triton X100), and centrifuged (2400 g, 20 min at 4°C). The supernatant was removed, the nuclei were resuspended in 500 µl of lysis buffer and then filtered in 70 µm and 40 µm filters consecutively. The nuclei were then collected by centrifuging the solution at 1000g for 5 min at 4°C. After washing with 950 µl 1×TAPS buffer (10 mM TAPS-NaOH, pH8.0, 5 mM MgCl2), the samples were centrifuged again at 1000 g for 5 min at 4°C. The supernatant was removed, leaving the nuclei suspended in approximately 10 µl of solution. Next, 1.5 µl of Tn5 transposase (Illumina FC-121-1030), 15 µl of Tagmentation buffer, and 13.5 µl of ddH20 were added to the solution. The reaction was incubated at 37°C for 30 min. The product was purified using a QIAGEN MinElute PCR Purification kit and then amplified using Phusion DNA polymerase. 1 µl of the product was used in 10 µl qPCR cocktail with Sybr Green. Cycle number X was determined as the cycle were the ¼ of the maximum signal was reached. Then we amplified the rest of the product in a PHusion (NEB) PCR system with X-2 cycles (10 to 15 cycles, 50 µl of reaction). Amplified libraries were purified with AMPure beads (Beckman Coulter), and library concentrations were determined using a Qubit. Sequencing was carried out in an Illumina HiSeqX (2×150 cycles) at the HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA).
GEO Accession
GSM4681512