GSM2807200: ddm1.rep1.mnase_seq; Arabidopsis thaliana; MNase-Seq
Approximately 1 g of pulverized flash frozen rosette leaf tissue from F4 generation plants was resuspended on ice in nuclei isolation buffer (0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 0.9% Triton X-100, 1 mM PMSF, 1X protease inhibitor cocktail) and allowed to thaw for 15 minutes. The suspension was then strained once through Miracloth (EMD-Millipore, cat. no. 475855) and the nuclear pellet was centrifuged in a swinging-bucket rotor at 2000g in pre-chilled 5 ml conical tubes. Buffer was carefully aspirated from the loose pellet, which was subsequently resuspended in TM2 (10 mM Tris–HCl, pH 8, 2 mM MgCl2) and washed twice in TM2. The pellet was then resuspended in pre-warmed MNase digestion buffer (50 mM Tris-HCl, pH 8, 5 mM CaCl2) and 25 units of MNase were added per 0.1 g initial starting material and incubated for 10 minutes at 37°C with periodic agitation; reactions were stopped with EGTA (20mM final concentration). This was followed by the addition of SDS to a final concentration of 0.625%, increasing the temperature to 75°C, addition of RNase A for 10 minutes, and the addition of proteinase-K for an additional 15 minutes. DNA was then extracted with phenol/chloroform, pelleted in 70% EtOH, then resuspended in TE pH 8.0. Verification of MNase digestion was carried out by fractionating one-tenth of the total reaction on a standard TAE gel and checking for a majority mononucleosomal fraction. Following removal of short DNA with beads, libraries were synthesized using total DNA (without PCR amplification) using the Nugen Ovation kit (Cat. no. 0319) following the manufacturer's protocol.
GEO Accession
GSM2807200