AA
Chromatin DNA samples were prepared from 0 and 5 dpa G. arboreum ovules and MNase-seq was carried out as previously reported (Webb et al. 2013; Ramachandran et al. 2017) with the following modifications: For each reaction, 5 g of nuclear chromatin DNA was suspended in 10 mM HEPES pH 7.4, 0.5 mM PMSF, and 0.5% NP40 on ice in a 500-L volume. MNase (Sigma) was added at a concentration of 10 U per ml. The mixture was incubated at 37 C for 15 minutes before addition of 1 L 1 M CaCl2 (final concentration 2 mM) to activate the MNase. Reactions were terminated by adding EGTA (1.5 mM), SDS (1%) and 20 g of proteinase K (Thermo Fisher Scientific) per ml of the reaction mixture, and incubated at 55C for a further 15 minutes. After phenol-chloroform-isoamylalcohol extraction and ethanol precipitation, the DNA pellet was dissolved in 50 L 1 mM Tris-HCl pH 8, 0.1mM EDTA and used directly for 1.5% agarose gel analysis and for making sequencing libraries. DNA sequencing was performed on the Illumina HiSeq4000 platform.