title: GSM4550565: ddm1_H2AX_Rep1; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: Nuclei isolation was performed using 4-5 week old leaves from WT Col-0 and 1st generation ddm1 as described previously. Freshly harvested tissues (0.3 g of tissue was used for each immunoprecipitation) were fixed with 1% formaldehyde for 15 min and the cross-linking reaction was stopped by the addition of 125 mM glycine. Crosslinked tissues were frozen and ground with a mortar and pestle in liquid nitrogen to obtain a fine powder. Ground tissues were resuspended in M1 buffer (10 mM sodium phosphate pH 7.0, 100 mM NaCl, 1 M hexylene glycol, 10 mM 2-mercaptoethanol, and protease inhibitor (Roche)), and the suspension was filtered through miracloth. Nuclei were precipitated by centrifugation and washed six times with M2 buffer (10 mM sodium phosphate pH 7.0, 100 mM NaCl, 1 M hexylene glycol, 10 mM MgCl2, 0.5% Triton X-100, 10 mM 2-mercaptoethanol, and protease inhibitor), and then further washed once with M3 buffer (10 mM sodium phosphate pH 7.0, 100 mM NaCl, 10 mM 2-mercaptoethanol, and protease inhibitor). Nuclei pellets were rinsed and resuspended in sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS, and protease inhibitor). Nuclei were sonicated with a Covaris E220 High Performance Focused Ultrasonicator for 15 min at 4˚C (Duty factor 5.0; PIP 140.0; Cycles per Burst 200) in 1 ml Covaris milliTUBE. After chromatin shearing, the debris was removed by centrifugation and the solutions containing chromatin fragments were diluted with three times the volume of ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS, and protease inhibitor). After dilution, protein A/G magnetic beads (50 µl for one gram of tissue; Thermo Fisher Scientific) were added to sheared chromatin and incubated at 4˚C for 1 hour. Pre-cleared samples were collected and incubated with anti-H3, anti-H2A.X.3/5, anti-H2A.W.6, anti-H1.1/1.2, or anti-H3K9me2 antibodies at 4˚C overnight with rotation. After incubation, samples were mixed with 30 µl of protein A/G magnetic beads, incubated at 4˚C for 3 hours with rotation, washed twice with low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), once with high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), once with LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% IGEPAL® CA-630, and 0.1% deoxycholic acid), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). Immunoprecipitated DNA was eluted by adding 500 µl of elution buffer (1% SDS and 0.1 M NaHCO3), incubated at 65˚C for 15 min, and mixed with 51 µl of reverse crosslink buffer (40 mM Tris-HCl pH 8.0, 0.2 M NaCl, 10 mM EDTA, 0.04 mg/ml proteinase K). The reaction mixture was then incubated at 45˚C for 3 hours and subsequently at 65˚C for 16 hours. After crosslink reversal, DNA was treated with 10 µg of RNaseA (Thermo Fisher Scientific), incubated at room temperature for 30 min, and purified with a MinElute PCR purification kit (Qiagen). For ChIP coupled with deep sequencing (ChIP-seq), sequencing libraries were prepared with an NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs) following the manufacturer's instructions. The libraries were sequenced with an Illumina Hiseq 2500 to generate single-end 50 bp reads. Samples were prepared from two independent biological replicates.
attributes.tag: GEO Accession
attributes.value: GSM4550565