title: GSM8297412: Col-0_input_Rep1; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: ~150 µL of mature pollens were collected and resuspended in ice-cold pollen nuclei buffer (1.3 mM H3BO3, 3.6 mM CaCl2·2H2O, 0.74 mM KH2PO4, 438 mM Sucrose, 7 mM MOPS, 0.83 mM MgSO4·7H2O, adjust pH to 6.0), and then transferred to a 2 ml tube containing 2 mm BashingBead, vortex at the 49 Hz for 3 min with homogenizer. Debris was removed by filtering the suspension through a 10 µm nylon mesh. Nuclei were precipitated by centrifugation and 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) containing 1% formaldehyde and fixed with 20 min, the cross-linking reaction was stopped by the addition of 125 mM glycine. Nuclei were precipitated by centrifugation and washed three 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 sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS, and protease inhibitor), and then sonicated with a sonicator (QSonica, Q800R3) for 30 min (20 s on/40 s off). After chromatin shearing, the debris was 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). For H3K9me2 ChIP, after dilution, protein G magnetic beads (Thermo Fisher Scientific) were added to sheared chromatin and incubated at 4˚C for 1 hour. Pre-cleared samples were collected and incubated with anti-H3K9me2 antibodies at 4˚C overnight with rotation. After incubation, samples were mixed with 30 µL of protein G magnetic beads, incubated at 4˚C for 3 hours with rotation. For SUVH6 ChIP, after dilution, the pre-cleared samples were collected and incubated with GFP-trap beads at 4˚C overnight with rotation. The beads then 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), twice 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% NP-40, and 0.1% deoxycholic acid), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). Immunoprecipitated DNA was incubated at 65 °C using 150 μL of elution buffer (1% SDS, 10 mM EDTA, 0.1 M NaHCO3) twice with shaking rigorously. After elution, the DNA-protein complex was reverse-crosslinked using 12 μL 5 M NaCl at 65 °C for 6 hours. 1 μL of Proteinase K, 10 μL of 0.5 M EDTA, 20 μL of 1 M Tris pH 6.5, 10 µg of RNaseA were added to digest the proteins and RNA. The DNA was then purified using phenol: chloroform and precipitated with sodium acetate, Glycogen and ethanol overnight at -20 °C. The precipitated DNA was collected and processed for the library construction. ChIP-Seq libraries were prepared using the VAHTS Universal DNA Library Prep Kit for Illumina V4 (Vazyme, ND610) following the manufacturer's instructions. CUT&Tag libraries were prepared using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903) following the manufacturer's instructions. The libraries were sequenced with an Illumina NovaSeq 6000 or Illumina Hiseq 3000 to generate pair-end 150 bp reads. Samples were prepared from three or two independent biological replicates.