GSM5597392: iMIP_rep2; Oryza sativa Japonica Group; ChIP-Seq
The fine powder was used for nuclei preparation and genomic DNA extraction following the procedures as described previously The genomic DNA was fragmented into sizes, ranging from 100-500 bp, using the water-based Biorupter (Diagnode), followed by DNA extraction and purification. Total 5μg fragmented genomic DNA was diluted in iM stabilization buffer (50mM Tris-AcOH, pH=5.5) for denaturing and re-association treatment using a PCR program as below: 95°C for 8 min，95°C for 30s (-0.5 °C /cycles, 129 cycles)，35°C hold on. The re-associated DNA was diluted with iM-IP incubation buffer (50mM Tris-AcOH, 1mM MgCl2, 130nM CaCl2, 1%BSA and Complete mini, pH=5.5), then incubated with 3μg iMab antibody (Ab01462-23.0, Kappa) for 4 h at 4℃. The antibody incubation reaction was incubated with 30 μl of washed protein G Dynalbeads (10004D, Invitrogen) for another 4 h at 4°C. The iMab-bound DNA was finally eluted with 200 μl elution buffer (0.1M NaHCO3 and 1% SDS) at 65°C for two times with 15 min each. Three biologically replicated iM-IPed DNA or Input/IgG-IPed control DNA was used for library preparation. All libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S). Libraries were finally quality controlled and sequenced using paired-end mode on Illumina NovaSeq platform.