GSM3109046: Mnase-seq_3h1_rep2; Arabidopsis thaliana; MNase-Seq
Nuclei were isolated from seedlings frozen in liquid nitrogen as described in (Chodavarapu, Feng et al. 2010) with following modifications: after resuspending in HBB, nuclei were applied to layer of HBB with 40% percoll (GE Healthcare), centrifuged at 1000g, 6 min, resuspended in HBB, applied to 40/75% percoll gradient, centrifuged at 400g, 40 min, collected and washed three times with HBC. The integrity of extracted nuclei was monitored using DAPI staining and fluorescence microscopy. The quantity of nuclei was measured by qPCR with primers targeting nuclear DNA. Digestion was performed by incubating nuclei suspended in DB buffer (16 mM Tris-HCl pH=7.6, 50 mM NaCl, 2.5 mM CaCl2, 0.01 mM PMSF, 1x Complete EDTA-free Protease Inhibitors (Roche)) with 1.5 μl of micrococcal nuclease (Thermo Fisher) and 2 μl of RNase A (Thermo Fisher) at 8°C for 90 min with gentle mixing. The reaction was stopped by adding equal volume of 2x Lysis buffer with EDTA (100 mM Tris-HCl pH=8, 200 mM NaCl, 50 mM EDTA, 1% SDS). The samples were lysed by incubation in 37°C for 60 min with shaking (1000 rpm). DNA was purified using phenol-chloroform extraction, precipitated with isopropanol and sodium acetate and resuspended in water. DNA was size selected by electrophoresis on 2% agarose gel with 1xTAE buffer with SYBR Gold (Invitrogen) stain. Mononucleosomal band was excised, frozen and squeezed by three cycles of spinning and rehydration on centrifuge column. DNA was purified and concentrated using Agencourt AMPure XP beads (Beckman Coulter). Barcoded libraries were synthesized from 100 ng of mononucleosomal DNA using Ion Xpress™ Plus gDNA Fragment Library Preparation Kit and Ion Xpress™ Barcode Adapters.
GEO Accession
GSM3109046