ChIP-seq Pol2
Plant seedlings were fixed with 1% formaldehyde solution in MS buffer (10 mM potassium phosphate, pH 7.0; 50 mM NaCl; 0.1M sucrose) at room temperature for 30 min in vacuum. After fixation, the seedlings were incubated at room temperature for 5 min under vacuum in MC buffer with 0.15 M glycine. Nuclei extraction was performed according to the part described in Hi-C protocol (Wang et al. 2015). Nuclei isolated from 1 g fixed material were used for 1 round of ChIP. The isolated nuclei were resuspended with 1 ml sonication buffer (10 mM potassium phosphate, pH 7.0; 0.1 mM NaCl; 0.5% Sarkosyl; 10 mM EDTA) and the chromatin was sheared by sonication with a CovarisTM S220 to achieve average fragment size around 300 bp. The sonicated sample was mixed wih 100 µl 10% Triton X-100, after which 50 µl was saved as input sample. The rest of sheared chromatin was mixed with equal volume of IP buffer (50mM Hepes, pH 7.5; 150mM NaCl; 5mM MgCl2; 10uM ZnSO4; 1% Triton X -100; 0.05% SDS), and incubated with anti-Pol2 (Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5), Abcam ab5408), or equally divided and incubated with anti-H3 (Abcam ab1791) and anti-H3K27me3 (Millipore, 07-449), respectively. After overnight incubation at 4 C°, 10 µl Protein A/G magnetic beads (Pierce) were further added and incubated at 4 C° for 2 hr. The beads were washed at 4 C° as following: 3 times with IP buffer, 1 time with IP buffer having 500 mM NaCl, and 1 time with LiCl buffer (0.25M LiCl; 1% NP-40; 1% deoxycholate; 1mM EDTA; 10mM Tris pH 8.0), with each wash lasted for 5 min. Chromatin retained on beads were incubated in 200 µl elution buffer (50 mM Tris, pH 8.0; 200 mM NaCl; 1% SDS; 10 mM EDTA) at 65 C° for 6 hr, followed by Proteinase K treatment at 37 C° for 1 hr. DNA extraction was performed with a standard phenol-chloroform extraction method, and all subsequent end repairing, A-tailing, adaptor ligation, library amplification steps were done by following a standard protocol (Illumina). The final library molecules were sequenced on Illumina HiSeq 2000 with 1x101 bp reads.