title: GSM3559817: WT_H3_2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: For γH2AX ChIP-seq, native ChIP method were performed (Bernatavichute et al., 2008). Briefly, MMS treated seedlings were harvested and ground to a fine powder in liquid nitrogen. Nuclei was extracted and washed with Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM HEPES (pH 7.4), 10 mM MgCl2, 0.5% Triton X-100, 5 mM DTT and 1X protease inhibitor (Roche)). Resuspended nuclei in 500μl 1X MNase Buffer (50mM Tris-HCl pH 7.6, 5mM CaCl2, 0.1 mM PMSF, 1X protease inhibitor (Roche)) and incubated them with RNase A at 37°C for 30 min. Then total volume was made to 1ml with 1X MNase Buffer and added 4.5 μl Micrococcal Nuclease (NEB, M0247S) for incubation at 37°C for 30min. Stopped the fragmentation process by adding EDTA (final 10 mM). Released nucleosomes by adding 0.1% SDS and rotated at 4°C for 3 hours. Then centrifuged and diluted the supernatant with MNase dilution buffer (0.1% Triton X-100, 50 mM NaCl, 0.1mM PMSF and 1X protease inhibitor). In advance, protein Dynabeads G (Invitrogen) beads should be incubated with antibodies (γH2AX and H3 antibodies are from ABclonal) in ChIP Dilution Buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1X protease inhibitor) at 4°C overnight. Beads-antibody complex was washed twice with 1ml MNase dilution buffer and added into diluted chromatins. The immunoprecipitation step was performed at 4°C for 5 hours. Then beads were washed twice with low salt wash buffer (50mM Tris-HCl pH 7.6, 10mM EDTA, 50mM NaCl, 0.1mM PMSF, 1X protease inhibitor), washed twice with middle salt wash buffer (50mM Tris-HCl pH 7.6, 10 mM EDTA, 100mM NaCl, 0.1mM PMSF, 1X protease inhibitor), washed once with high salt wash buffer (50 mM Tris-HCl pH 7.6, 10 mM EDTA, 150 mM NaCl, 0.1mM PMSF, 1X protease inhibitor), finally washed once with TE buffer (1 mM EDTA, 10 mM Tris-HCl pH8.0). Immune complexes were eluted by adding 200 μl elution buffer (0.1%SDS, 0.1M NaHCO3) at 65°C for 10 min, twice. Then treated with 2μl Proteinase K (10 mg/ml) for 1h at 45°C. Final DNA was recovered by Phenol/Chloroform extraction and ethanol precipitation. 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.
attributes.tag: GEO Accession
attributes.value: GSM3559817