title: GSM9682701: Wt_E2Fc_GFP_S6; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: Samples were ground in liquid nitrogen and resuspended in extraction buffer 1 (0.4 M sucrose, 10 mM tris-HCl pH 8, 10 mM MgCl2, 5 mM β-mercaptoethanol, and cOmplete Mini EDTA-free protease inhibitor cocktail). After filtration through 100-μm filters and centrifugation at 1500g for 10 min, pellets were resuspended and washed twice in extraction buffer 2 (0.25 M sucrose, 10 mM tris-HCl pH 8, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, protease inhibitors) for 5 min at 1000g. Nuclei were resuspended in nuclei lysis buffer (0.1% SDS, 50 mM tris-HCl pH 8, 10 mM EDTA pH 8). Chromatin was sonicated for 10 min using a Covaris S220 (peak power 175, cycles/burst 200, duty factor 20). Sonicated chromatin was immunoprecipitated overnight at 4°C with 2 μl anti-GFP antibody (Abcam ab290). Immunocomplexes were captured using 40 μl Dynabead protein A for 3 h at 4°C. Beads were washed nine times with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM tris-HCl pH 8, 167 mM NaCl, protease inhibitors). ChIP DNA was eluted with elution buffer (1% SDS, 0.1 M NaHCO3) at 65°C for 15 min (twice). Cross-links were reversed by addition of 5 M NaCl and overnight incubation at 65°C. Samples were treated with RNase A + T1 for 20 min and Proteinase K for 3 h at 50°C. DNA was extracted with phenol:chloroform:indole-3-acetic acid (25:24:1, pH 8) and precipitated with ethanol, NaOAc, and GlycoBlue. DNA was resuspended in nuclease-free water. Sequencing libraries were generated using a minimum of 2 ng ChIP DNA with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Library quality was assessed on an Agilent 2100 Bioanalyzer. Libraries were subjected to 75-bp high-throughput sequencing on an Illumina NextSeq 500.