GSM4506693: MNaseseq_eda16-OE_mock_rep2; Arabidopsis thaliana; MNase-Seq
flg22-treated and mock-treated seedlings were harvested onto lab tissue before freezing inmediatelly in liquid nitrogen. Frozen tissue was thoroughly ground to fine powder in liquid nitrogen using a pre-chilled pestle and mortar. RNA was extracted with the NucleoSpin® RNA kit (Macherey-Nagel) starting from ~100 mg of powder, following manufacturer's specifications. For MNase-seq experiments, 2 g of frozen powder were used for nuclei extraction with 10 mL of nuclei extraction buffer 1 (0.4 M sucrose, 10 mM Tris/HCl, pH 8.00, 10 mM MgCl2, 5 mM ß-mercapto-EtOH, 0.1 mM PMSF and Protease Inhibitor Mix P, 39103 Serva), filtering debris out through a 200 μm filter and centrifuging supernatant at 1000 g for 10 min at 4 °C. Nuclei pellet was washed in 5ml of nuclei extraction buffer 2 (25 mM Sucrose, 10 mM Tris/HCl pH 8.00, 10 mM MgCl2, 1% Triton X-100, 5 mM ß-mercapto-EtOH, 0.1 mM PMSF and Protease Inhibitor Mix P, 39103 Serva), mixed by vortex, filtered using a using a 60 μm filter and centrifuged at 1000 g for 10 min at 4 °C. Nuclei pellet was rinsed with Mnase buffer (10 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2, 0.15 mM Spermine and 0.5 mM Spermidine) and re-suspended in 250 μL of MNase buffer. DNA concentration was quantified with a NanoDrop, and samples were diluted to 400 ng/μL. 1 μl of 25 U/μL micrococcal nuclease (MNase) was added to 125 μL per sample and incubated at 37 °C for 10 minutes. To stop the reaction, 125 μL of Stop Buffer 2x (50 mM EDTA, 50 mM EGTA and 1% SDS) Tris/HCl pH 6.50 and 4 μL of proteinase K (stock 10 mg/ml) were added and incubated at 45 °C for 1h. Samples were purified with a QIAquick PCR Purification Kit (Qiagen) and eluted with 15 μL water. Eluate was loaded onto a 1% agarose gel (without loading buffer dye) and the lowest band (mono-nucleosomal DNA) was excised and gel-purified with QIAquick® Gel Extraction Kit (Qiagen). RNA library prep was carried out with a #E7420 S/L NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (New England Biolabs) following manufacturer's instructions. Agencourt® AMPure® XP Beads (#A63881, Beckman Coulter, Inc.) magnetic beads were used for RNA purification. Genomic libraries for MNase-seq were prepared starting from 50 ng of DNA per sample. DNA library prep was carried out with a NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (E7645S/L, NEB) following manufacturer's instructions. Agencourt® AMPure® XP Beads (#A63881, Beckman Coulter, Inc.) magnetic beads were used for DNA purification.
GEO Accession
GSM4506693