title: GSM6205330: minu, rep2; Arabidopsis thaliana; MNase-Seq
library_construction_protocol: 0.35gr of inflorescences from Col-0, minu1-2 minu2-1, and tpf1-1 tpf2-1 were ground and resuspended in 20ml of Isolation Buffer 1 (20mM Tris pH8, 0.3 M sucrose, 5mM MgCl2, 0.2% Triton X100- 5mM BME 14.3M, 35% glycerol, 5ul 0.2M PMSF in 10ml, Complete mini –EDTA). After filtering through 1 layer of miracloth samples were spun at 3200rpm 4C for 20 min. The pellet was resuspended in 1ml of HBB buffer (25 mm Tris–HCl, pH 7.6, 0.44 M sucrose, 10 Mm MgCl2, 0.1% Triton-X and 10 mm β-mercaptoethanol, 5ul 0.2M PMSF in 10ml. Complete mini –EDTA) and spun at 3200rpm 4C for 10 min. The pellet was resuspended in 1ml of MNB buffer (10% sucrose, 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, and 1 mM CaCl2) and spun at 3200 rpm 4C for 10 min. The pellet was resuspended in 900 ul of MNB buffer and 180 ul aliquots were digested with different amounts of Micrococcal Nuclease (N3755, Sigma) (0, 1, 3, 5 and 8U) followed 10 min incubation at 37C, quickly mixing samples every 3 min. Reactions were stopped by addition of 20 μL of 0.5 mM EDTA, and 4ul of DNAse-Free RNAse (10mg/ml) were added followed by incubation at 37C for 1h. 1ul proteinase K (20mg/ml) was added followed by incubation at 45C for 1h. DNA was extracted with Phenol:Chloroform:isoamyl followed by chloroform extraction and precipitation with NaOAc/glycogen/ethanol at −20 °C overnight. Samples were centrifuged at 13,000 × g for 15 min at 4 °C, washed with 70% ethanol and resuspended in 30ul elution buffer. Samples were run in a 2% agarose gel to recover mononucleosomal DNA (aprox. 150bp) from digestions that presented 20% di-nucleosome and 80% of mono-nucleosome. Gel bands were cut and purified with Zymo gel extraction kit. Libraries for sequencing were prepared at BGI following the BGISEQ-500 ChIP-Seq library preparation protocol. Libraries for sequencing were prepared at BGI following the BGISEQ-500 ChIP-Seq library preparation protocol.