title: GSM9241043: Wild-type IP chromatin, biol rep 2; Petunia x hybrida; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation on two biological replicates for WT, star and wico petals at stage 8 was performed as follows: petals were flash-frozen and ground in liquid nitrogen, and ground tissue was resuspended into 10 mL fixation buffer (10 mM Hepes pH7.6, 0.5 M sucrose, 5 mM KCl, 5 mM MgCl2, 5 mM EDTA pH8, Complete Protease Inhibitor Cocktail (Merck), 14 mM 2-mercaptoethanol) and a double cross-linking was performed at room temperature (1 hour with disuccinimidyl glutarate at 2.5 mM with gentle shaking, and 5 minutes with formaldehyde 1% (v/v)). Cross-linking was stopped by adding glycine at 200 mM and samples were put directly on ice. Cells were lysed with a 40 mL-Dounce tissue grinder, Triton X-100 was added at 0.6% (w/v) and the lysate was filtered subsequently through 100 µm and 40 µm nylon meshes to recover nuclei. Nuclei were pelleted for 10 minutes at 3,000 g at 4°C, and the pellet was resuspended in 300 µL of cold nuclear isolation buffer (i.e. fixation buffer without 2-mercaptoethanol), carefully deposited on 600 µL of a 15% Percoll solution (15 % (v/v) Percoll, 10 mM Hepes pH8, 0.5 M sucrose, 5 mM KCl, 5 mM MgCl2, 5 mM EDTA pH8) and centrifuged for 5 minutes at 2,000 g at 4°C. The pellet was resuspended into 900 µL of cold nuclear lysis buffer (50 mM Tris-HCl pH7.5, 0.1% (w/v) SDS, 10 mM EDTA pH8) to lyse the nuclei, and chromatin was sonicated twice for 15 minutes with a Covaris S220 sonicator (peak power 105, Duty factor 5, Cycles/Burst 200 for 900s). For each sample, 25 µL of magnetic protein-A Dynabeads and 25 µL of magnetic protein-G Dynabeads (Invitrogen) were washed twice with 100 µL of cold ChIP dilution buffer (15 mM Tris-HCl pH7.5, 150 mM NaCl, 1% (w/v) Triton X-100, 1 mM EDTA pH8) using a magnetic rack. Beads were mixed with 2.5 µg of anti-PhDEF antibody and 1.8 mL of cold ChIP dilution buffer, and incubated for 2 hours at 4°C on a rotating wheel. Sonicated chromatin was centrifuged for 5 minutes at 15,000 g at 15°C, and 25 µL of supernatant (for wt samples) or 100 µL of supernatant (for star and wico samples) was added to the mix of beads and antibody, and incubated overnight at 4°C on a rotating wheel. Beads were washed twice (one quick wash and one long wash with 15 minutes incubation on a rotating wheel) with each of the following buffers: low salt wash buffer (0.1% (w/v) SDS, 1% (w/v) Triton X-100, 2 mM EDTA pH8, 20 mM Tris-HCl pH8, 150 mM NaCl), high salt wash buffer (0.1% (w/v) SDS, 1% (w/v) Triton X-100, 2 mM EDTA pH8, 20 mM Tris-HCl pH8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% (v/v) NP40/Igepal,1% (w/v) deoxycholate, 1 mM EDTA pH8, 20 mM Tris-HCl pH8) and TE buffer. Elution was performed twice with 250 µL of elution buffer (0.1 M NaHCO3, 1% (w/v) SDS) at 65°C. IP and input samples were decrosslinked overnight at 65°C by adding NaCl at 200 mM, then incubating for 2 h at 42°C with 20 µg proteinase K in 10 mM EDTA pH8 and 40 mM Tris-HCl pH6.5. DNA was purified with phenol:chloroform:isoamyl alcohol (25:24:1) followed by chloroform:isoamyl alcohol (24:1), precipitated with ethanol at -20°C and the pellet was washed with ethanol 70 %. The dry pellet was recovered in 50 µL TE. The quality of IP and INPUT samples was assessed with Tapestation 4150 HS D5000 (Agilent). Libraries were prepared with MicroPlex Library Preparation Kit v3 (Diagenode) and around 1 ng of each library was PCR-amplified (12 cycles), following Diagenode's recommendations. Libraries were analyzed Tapestation 4150 HS D5000 and quantified with Qubit 4.0 with the Qubit dsDNA HS Assay Kit (Thermofisher), then mixed at equimolar ratio for sequencing with an Illumina NextSeq 500 (paired-end, 2x76 bp, dual indexing).