title: GSM5514278: EcTopoI ChIP-Seq, no treatment, replicate 2; Escherichia coli; ChIP-Seq
library_construction_protocol: Exponentially growing culture (OD600=0.6) in LB was crosslinked with 1% formaldehyde. Formaldehyde was quenched by incubation with 0.125 M glycine. Cells were harvested by centrifugation and washed three times with 10 mL ice-cold PBS and finally resuspended in 1mL of FA lysis buffer (50 mM HEPES-NaOH pH 7.5, 1 mM EDTA, 0.1% Deoxycholate, 0.1 % SDS, 1% Triton X-100, 150 mM NaCl). Then, protease inhibitors cocktail (cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific) were added. To disrupt cells and shear DNA, cells were sonicated in a 1.5 mL tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysates were clarified by centrifugation, and 900 µL of the lysate were diluted with 1ml of TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) and mixed with 80 µL of ANTI-FLAG® M2 affinity gel (Sigma-Aldrich). Immunoprecipitation was performed at room temperature on a rotator for 1.5 h. Then affinity resin was washed consecutively with 1 mL of the following solutions: TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl); twice with TESS buffer (10 mM Tris-HCl pH 7.5, 250 mM NaCl, 1 mM EDTA, 0.1% Tween-20, 0.05% SDS); and TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA). For proteolysis and de-crosslinking after the washing procedure, affinity resin was diluted with 200 µL of TES buffer, proteinase K (Sigma-Aldrich) was added up to 0.5 mg/mL and samples were incubated at 55°C for 4 h. Supernatant was collected by centrifugation (2000 g, 2 min, 25°C) and IP-DNA was finally purified from it using AMPure XP magnetic beads (Beckman Coulter). Sequencing libraries were prepared using TruSeq kit (Illumina). Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 2. For Input DNA (mock sample) 100 µL of lysate was treated and de-crosslinked by incubation with proteinase K (Thermo-Fisher Scientific) for 4 h at 55°C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). For EcTopoI immunoprecipitation (IP sample), 900 µL of the remaining lysate were diluted with 1ml of TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) and mixed with 80 µL of ANTI-FLAG® M2 affinity gel (Sigma-Aldrich). Immunoprecipitation was performed at room temperature on a rotator for 1.5 h. Then affinity resin was washed consecutively with 1 mL  of the following solutions: TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl); twice with TESS buffer (10 mM Tris-HCl pH 7.5, 250 mM NaCl, 1 mM EDTA, 0.1% Tween-20, 0.05% SDS); and TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA). For proteolysis and de-crosslinking after the washing procedure, affinity resin was diluted with 200 µL of TES buffer, proteinase K (Sigma-Aldrich) was added up to 0.5 mg/mL and samples were incubated at 55°C for 4 h. Supernatant was collected by centrifugation (2000 g, 2 min, 25°C) and IP-DNA was finally purified from it using AMPure XP magnetic beads (Beckman Coulter). Sequencing libraries were prepared using TruSeq kit (Illumina). Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Libraries preparation and sequencing were performed at Skoltech Sequencing Core Facilities. ChIP-Seq was performed in triplicate.