Maize B73 Seedling Root MNase-seq
Ten grams of tissue were ground under liquid nitrogen with a mortar and pestle and cross-linked by stirring for 10 min in 100 mL ice-cold fixation buffer (15 mM PIPES-NaOH, pH 6.8, 0.32 mM sorbitol, 80 mM KCl, 20 mM NaCl, 0.5 mM EGTA, 2mM EDTA, 1mM DTT, 0.15 mM spermine, and 0.5 mM spermidine) containing 1% formaldehyde. Fixation was stopped by adding glycine to 125 mM. Nuclei were isolated by adding Triton X-100 to 1% and stirring for 10 minutes. The suspension was filtered through one layer of Miracloth (Calbiochem) and placed in 50 mL centrifuge tubes. In 50 mL centrifuge tubes, 35 mL nuclear suspensions were underplayed with 15 mL of Percoll cushion composed of 50% Percoll (GE) in BFA. Nuclei suspensions were centrifuged at 3000g for 15 min at 4°C. The nuclei at the Percoll interface were transferred to a 50 mL tube and diluted two-fold with MNase digestion buffer (“MDB”, containing 50 mM Tris-HCl, pH 7.5, 320 mM sucrose, 4 mM MgCl2, and 1mM CaCl2). Nuclei suspensions were centrifuged at 2000g for 10 minutes at 4°C, and nuclei pellets were resuspended in 2.5 mL MDB. Nuclei were aliquoted into 500 mL aliquots, flash frozen in liquid nitrogen, and stored at -80°C until use. Nuclei were thawed at room temperature, digested by adding MNase to 10U/mL (light) or 100 U/mL (heavy), and incubating at room temperature for 5 minutes. Digestions were stopped with 10 mM EGTA. Nuclei were de-cross-linked by incubating overnight at 65°C in the presence of 10% SDS and 100 ug/mL Proteinase K. DNA was extracted by phenol-chloroform extraction followed by EtOH precipitation. Digested DNA was resuspended in 40 ug/mL RNase A and electrophoresed in a 1% agarose gel. DNA fragments under 200 bp were excised and gel extracted following ethidium bromide staining with the Qiaex II gel extraction kit (Qiagen) following the manufacturer’s instructions. Following nucleus isolation and digestion, gel-extracted DNA was used to prepare sequencing libraries using the NEBNext® Ultra DNA Library Prep Kit for Illumina (NEB) using manufacturer instructions. Indexed libraries were pooled and sequenced on 8 Illumina HiSeq 2500 lanes with paired-end 50-cycle sequencing.