GSM7091095: Col0_rep1_g2_MNase-seq; Arabidopsis thaliana; MNase-Seq
1.5g samples of 12-day-old seedlings were ground into powder in liquid nitrogen and homogenizated in 20 ml Honda Buffer (0.44 M Sucrose, 1.25 % Ficoll, 2.5 % Dextran T40, 20 mM HEPES-KOH pH 7.4, 10 mM MgCl2, 0.5 % Triton X-100, 5 mM DTT, EDTA-free cOmplete). After filtration with two layers of Miracloth (Merck Millipore, 475855), the nuclei were centrifuged and washed twice with 10 ml Honda Buffer, and were eventually resuspended with 900 ul of MNase digestion buffer (20 mM Tris-Cl pH 8, 5 mM NaCl, 2.5 mM CaCl2). The isolated chromatin was digested with gradient concentration of Micrococcal Nuclease (Takara, 2910 A) for 10 min at 37 °C to obtain mostly mono-nucleosomes. The digestion was stopped with 10 mM EDTA and treated with lysis solution (1% SDS, 50 mM Tris-HCl pH 8, 20 mM EDTA) and 20 mg/ml Proteinase K at 37 °C overnight. DNA was purified with phenol-chloroform-isoamyl alcohol, and was precipitated with salts and ethanol followed by treatment with 10 mg/ml RNase A. The digestion efficiency of different enzyme gradient was examined on a 2 % agarose gel through electrophoresis. The reaction generating 80% of mono-nucleosome was chosen, where the DNA band corresponding to ~150 bp was cut from gel and purified with a the QIAquick gel extraction kit (Qiagen, 28115). The purified DNA band corresponding to ~150 bp were sent to Novogene (Tianjin, China) for library preparation and deep sequencing.