title: GSM3891763: G4-ChIP-seq_K++PEG_rep1; Oryza sativa; ChIP-Seq
library_construction_protocol: The purified nuclei were fragmented into sizes ranging from 100-500 bp in sonication buffer (50 mM Tris-HCl + 10 mM EDTA + 1% SDS) using the water-based Biorupter (Diagnode) followed by reverse cross-linking at 65 ℃ overnight，which 5M NaCl was added to a final concentration 0.2M. The reverse cross-linked DNA was purified by phenol:chloroform extraction and ethanol precipitation.Total 5 μg fragmented genomic DNA was diluted in G4 stabilization buffer (GSB) (150 mM KCl + 40% PEG200 + 10 mM Tris-HCl, pH=7.5), and denatured at 95 ℃ for 5 min followed by reassociation with the temperature slowly dropped down to RT. The reassociated DNA was diluted with G4-ChIP incubation buffer (GCB) (50 mM HEPES + 150 mM KCl+ 1 mM MgCl2+ 130 nM CaCl2+1 % BSA+ 40% PEG200 + Complete mini, pH 7.5). 3 μg purified BG4-FLAG recombinant protein was added and incubated at 4 ℃ for 4 h with constant rotation. 3 μg anti-FLAG antibody (D110005, BBI) was then added and incubated at 4 ℃ for additional 4 h. 30 μl protein G Dynalbeads (10004D, Invitrogen) was washed with incubation buffer for three times, and added to the reaction for incubation at 4℃ for another 4 h. After protein G beads incubation, the beads were collected and washed with washing buffer (10 mM Tris-HCl + 150 mM KCl + 1% Tween20) for three times. The BG4-bound DNA was eluted with 200 μl elution buffer (0.1 M NaHCO3 + 1% SDS) at 65 ℃ for two times with 15 min each. ChIPed and corresponding input DNA was recovered and dissolved in 20 EB (10 mM Tris-HCl, pH=8.0). For BG4-ChIP-seq assay, the BG4-ChIPed DNA was used for library preparation for Illumina sequencing followed by data analysis.