Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: The protocol for native chromatin immunoprecipitation was adapted from Bernatavichute et al., 2008. Leaf 4 material of 26 day-old wild-type and PEG1 plants was harvested, frozen in liquid nitrogen and ground to powder (1.5 g). Plant tissue was resuspended in 15 ml of HBM buffer (25 mM Tris-Cl pH 7.5, 440 mM sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM beta-mercaptoethanol, 2 mM spermine, 1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin), homogenized and filtered twice through Miracloth (Calbiochem). After spinning at 3000 rpm for 5 min (4°C, SS-34, Sorvall), the pellet was resuspended in 5 ml of NIB buffer (20 mM Tris-Cl pH 7.5, 250 mM sucrose, 5 mM MgCl2, 5 mM KCl, 0.1% Triton-X, 10 mM beta-mercaptoethanol, 1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin), applied to a 15/50% Percoll (GE Healthcare) gradient in NIB and spun 2000 rpm for 20 min (4 C, SS-34, Sorvall). Isolated nuclei were washed three times in NIB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl pH 7.5, 440 mM sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM beta-mercaptoethanol, 20% glycerol). Nuclei from each preparation were diluted 2-fold in digestion buffer (20 mM Tris-Cl pH 7.5, 0.22 M sucrose, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 5 mM Na butyrate), spun 600 g for 5 min (4°C, Eppendorf, 5415R) and resuspended in 0.25 ml digestion buffer for Micrococcal Nuclease (30 U, Affymetrix) digestion for 10 minutes at 37 °C stopped with 10 mM EDTA. Mononucleosomes were released by treating nuclei with 0.1% Triton-X for 2 hours in the cold, and then pelleting the debris by centrifuging at 10000 rpm for 5 min (4°C, Eppendorf, 5415R). The supernatants were diluted to 0.5ml in incubation buffer (20 mM Tris-Cl pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.1% Triton, 20 mM Na butyrate, 0.1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin) and overnight incubated in the cold with and without 2.5 ug of the anti-H3K4m3 antibody (#07-473, Millipore). 50 ul of Dynabeads Protein G (Invitrogen) were applied, incubated for 3 hours in the cold and washed three times with 500 ul wash buffer (50 mM Tris-Cl pH 7.5, 10 mM EDTA, 5mM Na butyrate, 0.1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin) with increasing concentrations of NaCl (50 mM, 100 mM and 150 mM), subsequently. Final wash was done in TE buffer (10 mM TrisCl pH8, 500 mM NaCl, 1 mM EDTA) and immunocomplexes were eluted and reverse crosslinked by incubation overnight at 65°C in 0.25 ml 10 mM TrisCl pH8, 1 mM EDTA, 0.5 M NaCl, 1% SDS + 1 µl RNAse A (100 mg/ml). Eluates were incubated with 100 µg Proteinase K for 2 h at 42°C and the DNA extracted by phenol/chloroform/IAA followed by purification using Qiaquick PCR purification kit and DNA quantification by the Quant-iT dsDNA High Sensitivity kit (Invitrogen). Standard Illumina Protocol