Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Lysates from sonicated ceratin sigma factors-DNA complexes were isolated with specific antibodies. ChIP-exo experiment was performed following the procedures: to identify each sigma factor binding maps in vivo, we isolated the DNA bound to each sigma factor from formaldehyde cross-linked Salmonellaonella enterica serovar Typhimurium LT2 cells by chromatin immunoprecipitation (ChIP) with the specific antibodies that specifically recognize subunits of RNA polymerase (cat# WP002, neoclone), sigma factor 70 (cat# WP004, neoclone), sigma factor 54 (cat# W0005,neoclone), and Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) followed by stringent washings as described previously. ChIP materials (chromatin-beads) were used to perform on-bead enzymatic reactions of the ChIP-exo method. 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 65oC. RNAs- and Proteins-removed DNA samples were used to perform primer extension and second adaptor ligation with following modifications. The DNA samples incubated for primer extension as described previously [12] 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. Each modified step was also performed in accordance with the manufacturer's instructions. ChIP-exo experiments were performed in biological duplicate.