GSM3559813: WT_gamma_2; Arabidopsis thaliana; ChIP-Seq
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.
GEO Accession
GSM3559813