title: GSM5896540: NIP MH-seq Rep1; Oryza sativa Japonica Group; DNase-Hypersensitivity
library_construction_protocol: MH-seq: Two-week-old seedlings from each variety were collected for cross-linking using 1% formaldehyde in HEPES buffer pH=8.0 (20 mM HEPES, 1 mM EDTA, 100 mM NaCl and 1mM PMSF). The cross-linked samples were ground into a fine powder in liquid nitrogen for the preparation of nuclei and chromatin for the downstream experiments. MH-seq: Nuclei isolation and purification was conducted as previously described (Zhang and Jiang 2018). The nuclei pellet was re-suspended using 1.0 ml of MNase digestion buffer, and equally divided into 200 μl each aliquot. The aliquoted nuclei were trimmed at 37 ℃ for 10 min using a series of MNase. After stopping enzymatic digestion using 0.5 M EDTA (pH 8.0), each MNase cleaved nuclei were reversely cross-linked at 65 ℃ overnight. After removal of RNA and protein contamination using sequential treatment with RNase A at 37 ℃ for 30 min and proteinase K at 55 ℃ for 2 h. DNA was precipitated using cold ethanol after phenol/chloroform purification. The DNA pellet was dissolved using 20 μl EB (10 mM Tris-HCl, pH 8.0). After running 12% PAGE in 1xTBE buffer, 40-100 bp small sized DNA fragments were recovered from the purified DNA for MNase hypersensitive sequencing (MH-seq) library preparation. MH-seq library was prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645S).