title: GSM5110920: Seed tissue - MH-seq_rep1; Glycine max; MNase-Seq
library_construction_protocol: The collected material was ground into fine powder in liquid nitrogen. Then the powder (0.5g for leaf and 1 g for flower, seed and root) was fixed with 1% of final concentration of formaldehyde for 10 min and followed by neutralization with 0.125 M glycine for 5 min. Next, the nuclei were prepared following previous protocols. The purified nuclei were suspended in 0.8 ml MNase digestion buffer (MNB, 10% sucrose, 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, and 1 mM CaCl2) and divided into 4 of 1.5 ml eppendorf tubes. The aliquoted nuclei were cleaved at 37°C for 10 mins by using a series of MNase (N3755-50UN, Sigma) in 0, 0.01, 0.03 and 0.1 unit, respectively. After terminating digestion with 10 mM EDTA, the cleaved nuclei with 200 mM NaCl were incubated at 65 °C for 4h in order to reverse cross-linking. The DNA was extracted by adding 400 ul CTAB and incubated at 65°C for 15 min. The subsequent step was performed by following CTAB method. The extracted DNA was separated by running 2% of agarose gel in 1x TAE buffer. We recovered the DNA fragment with size less than 100 bp to prepare MH-seq library. Illumina library following standard Illumina library preparation procedures.