title: GSM3443311: WT; Arabidopsis thaliana; OTHER
library_construction_protocol: ssDRIP was performed as described previously (Xu et al., 2017) with some modifications. Briefly, 14-day-old seedlings (3g) were ground into fine powder in liquid N2 and resuspended in 30 ml pre-cooled Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM HEPES KOH, pH 7.4, 10 mM MgCl2, 0.5% Triton X-100). After filtering through two layers of Miracloth and centrifuging at 2000 g for 15 min at 4°C, nuclei were washed 2–3 times with NRBT buffer (20 mM Tris-HCl pH 7.5, 25% Glycerol, 2.5 mM MgCl2, 0.2% Triton X-100). Pellet was resuspended in 2 ml TE buffer (1 mM EDTA pH8.0, 10 mM Tris‐HCl pH8.0) with 0.5 % SDS and 0.33 mg/ml Proteinase K and was incubated constant temperature shaker at 37°C, 400 rpm, overnight. DNA was recovered with phenol:chloroform extraction and purified genomic DNA was digested with MseI, MboI, DdeI and NlaIII (NEB) for fragmentation. For S9.6 immunoprecipitation, 4 μg fragmented DNA (DNA concentration was measured with Qubit dsDNA kit) was incubated with 10 μg S9.6 antibody in DRIP Binding Buffer (10 mM NaPO4 pH 7.0, 0.14 M NaCl, 0.05% Triton X-100) at 4°C overnight. DNA-antibody complexes were incubated with Protein G beads (Invitrogen) at 4°C for 4 hours with rotation then washed 4 times by DRIP Binding Buffer at room temperature. DNA was eluted with Elution Buffer (50 mM Tris pH 8.0, 10 mM EDTA), together with 200 μg Proteinase K incubation at 55°C for 1 hour, 1000 rpm. Finally, DRIPed DNA was recovered with phenol:chloroform extraction. 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.