GSM8269028: WT-MNase-R2; Arabidopsis thaliana; MNase-Seq
For nuclear extraction, 2-week-old Arabidopsis seedlings without roots were ground in liquid nitrogen and resuspended in lysis buffer [50 mM HEPES, pH7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 10% glycerol, 5 mM b-mercaptoethanol and protease inhibitor cocktail. After lysis for about half an hour, the lysis mixture was filtered through a 40-lm cell strainer. The filtered plant extracts were centrifuged for 20 min at 3200 g to precipitate nuclei. Isolated nuclei were washed once with HBB buffer (25 mM Tris–HCl, pH 7.5, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton-X and 10 mM b-mercaptoethanol) and centrifuge at 1500g for 10 min at 4°C. Once with HBC buffer (20 mM Tris–HCl, pH 7.5, 352 mM sucrose, 8 mM MgCl2, 0.08% Triton-X, 8 mM b-mercaptoethanol and 20% glycerol) and centrifuge at 1500g for 10 min at 4°C. The obtained nuclei were resuspended with 100 ul of HBC buffer and flash frozen in liquid nitrogen. Two ng of DNA was used for library construction.  Libraries were constructed by the VAHTSTM Universal DNA Library Prep Kit for Illumina® V3 (ND607), VAHTSTM DNA Adapters set3 - set6 for Illumina® (N805) and VAHTS® DNA Clean Beads (N411-02, Vazyme) according to the manufacturer's protocol. protocol. Illumina Novaseq was used for high-throughput sequencing of the ChIP-seq library.