title: CLF-H3K4m2
organism: Chamaelirium luteum
description: Protocols: The perennial Chamaelirium luteum plants were grown at IPK Gatersleben (Germany) in a greenhouse: 16 h light (from 6 AM to 10 PM), day temperature 16 °C, night temperature 12 °C. Chromatin isolated from 0.33 g of Cha. luteum developing inflorescence with unopened floral buds was used for the ChIP experiment of the histone marks.   The perennial Chamaelirium luteum plants were grown at IPK Gatersleben (Germany) in a greenhouse: 16 h light (from 6 AM to 10 PM), day temperature 16 °C, night temperature 12 °C. Chromatin isolated from 0.33 g of Cha. luteum developing inflorescence with unopened floral buds was used for the ChIP experiment of the histone marks. 0.65 g of C. luteum flower and 1.0 g of Secale cereale (inbred line Lo7) leaf tissue were ground with liquid nitrogen and homogenized separately in 10 ml nuclei isolation buffer (1 M sucrose, 5 mM KCl, 5 mM MgCl2, 60 mM HEPES pH 8.0, 5 mM EDTA, 0.6% Triton X-100, 0.4 mM PMSF, 1 µM pepstatin A, cOmplete protease inhibitor cocktail (Roche)). Nuclei fixation was performed in 1% PFA/ nuclei isolation buffer at RT, 12 rpm for 10 min and terminated by adding glycine to a final concentration of 130 mM. The nuclei suspension was filtrated through Miracloth (Millipore) twice and a 50-µm CellTrics filter (Sysmex) once and centrifuged at 4°C, 3,000 ×g for 10 min. The nuclei pellet was resuspended in 1 ml extraction buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 µM pepstatin A, cOmplete protease inhibitor cocktail), followed by centrifugation at 4°C, 12,000 ×g for 10 min. After removing the supernatant, nuclei were resuspended in 150 µl of nuclei lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 0.1 mM PMSF, 1 µM pepstatin A, cOmplete protease inhibitor cocktail). Chromatins were sonicated for 14 cycles of 30 s ON, 30 s OFF at high power, in a Bioruptor (Diagenode), followed by an addition of 100 µl ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 1 mM EDTA, cOmplete protease inhibitor cocktail), and continued sonication to a total of 31 cycles under the same setting. The sonicated samples were diluted 10 times with ChIP dilution buffer, centrifuged at 4°C, 13,000 ×g for 5 min, and the supernatant of each samples were transferred to new tubes. To dilute the high proportion of the putative C. luteum centromeric repeat, sonicated chromatin of C. luteum and S. cereale was mixed in a 1:3 ratio. The mixed chromatins were incubated with the ClCENH3 antibody (10 mg/ml) to a final 1:500 dilution at 4°C by shaking at 14 rpm for 12 h. DynabeadsTM Protein A (Invitrogen) in ChIP dilution buffer, corresponding to 0.1× volume of the chromatin solution, was added to the antibody-prebound chromatins and incubated at 4°C by shaking at 14 rpm for 1.5 h. The collected beads were then washed twice in low salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris-HCl pH 8.0), followed by three washes in high salt buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), and another two washes in TE buffer at 4°C by shaking at 14 rpm for 5 min. The bead-bound chromatin was purified by using iPure kit v2 (Diagenode) following the manual and quantified using QubitTM dsDNA HS Assay kit (Invitrogen). Genomic DNA of C. luteum was extracted from leaf tissue using the DNeasy Plant Mini kit (Qiagen, Germany)  Genomic DNA of C. luteum was extracted from leaf tissue using the DNeasy Plant Mini kit (Qiagen, Germany)