title: GSM7075985: Col_Input_Set8; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: 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 a sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS, and protease inhibitor) and 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 were 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 with rotation. Pre-cleared samples were collected and incubated with 5 µg of anti-H3 (Abcam, ab1791), anti-H2A.X.3/5, anti-H2A.13, anti-H2A.2, anti-H2A.Z.9, anti-H2A.Z.11, anti-H2A.W.6, anti-H2A.W.7, anti-H1, anti-H3K36me3 (Abcam, ab9050), anti-H3K27me3 (Millipore, 07-449), anti-H3K4me3 (Abcam, ab8580), anti-H3K4me1 (Abcam, ab8895), anti-H3K27me1 (Millipore 17-643), anti-H4K20me1 (Abcam, ab9051), anti-H3K9me1 (Abcam, ab8896) or anti-H3K9me2 (Abcam, ab1220) 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®