GSM5526084: ArcA_4D6; Escherichia coli K-12; OTHER
Lysates were clarified from sonicated TFs-DNA complexes, and then were isolated with custom anti-ArcA monoclonal antibodies. The experiments were performed following the procedures: to identify the binding maps in vivo, we isolated the DNA bound to ArcA from formaldehyde cross-linked E. coli cells by chromatin immunoprecipitation (ChIP) with the custom anti-ArcA monoclonal antibodies that specifically recognize ArcA, and Dynabeads Pan Mouse IgG magnetic beads (Invitrogen). ChIP materials (chromatin-beads) were used to perform on-bead enzymatic reactions. Briefly, the sheared DNA of chromatin-beads was repaired by the NEBNext End Repair Module (New England Biolabs) followed by the addition of a single dA overhang and ligation of the first adaptor (5'-phosphorylated) using dA-Tailing Module (New England Biolabs) and NEBNext Quick Ligation Module (New England Biolabs), respectively. Nick repair was performed by using PreCR Repair Mix (New England Biolabs). Lambda exonuclease- and RecJf exonuclease-treated chromatin was eluted from the beads and the protein-DNA cross-link was reversed by overnight incubation at 65 degree. RNAs- and Proteins-removed DNA samples were used to perform primer extension and second adaptor ligation. The DNA samples incubated for primer extension were treated with dA-Tailing Module (New England Biolabs) and NEBNext Quick Ligation Module (New England Biolabs) for second adaptor ligation. The DNA sample purified by GeneRead Size Selection Kit (Qiagen) was enriched by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The amplified DNA samples were purified again by GeneRead Size Selection Kit (Qiagen) and quantified using Qubit dsDNA HS Assay Kit (Life Technologies). Quality of the DNA sample was checked by running Agilent High Sensitivity DNA Kit using Agilent 2100 Bioanalyzer (Agilent) before sequenced using HiSeq (Illumina) in accordance with the manufacturer's instructions. The experiments were performed in biological duplicate.