Nuclei were isolated from 12-day-old Arabidopsis Col-0 seedling, followed by SDS/proteinase K treatment at 37°C overnight. Genome DNA was extracted by the phenol-chloroform method and precipitated with isopropanol. DNA fragmentation was performed overnight using endonucleases (SspI, EcoRI, HindIII, DdeI, MspI, and RsaI); the negative control was treated with RNase H (New England Biolabs) overnight. DRIP was performed as described in Ginno et al., 2012 and Sun et al., 2013. Briefly, 4ug of DNA was bound with 10 ug of S9.6 antibody in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4˚C. 50 ul Protein G sepharose beads were added for 4 hours. Bound beads were washed 4 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) for 45 min at 55˚C. DNA was purified and sonoicated to 150 bp fragments . The first adapter was ligated to the 3’ end of the ssDNA using Adaptase (Swift Biosciences) via a highly efficient, proprietary reaction that only tails 3’ ends of ssDNA and ligates the first truncated adapter to 3’ ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequence-independent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter to the 5’ ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified.
GEO Accession
GSM2525603