GSM7025988: Topo-Seq, E. coli DY330 parC-SPA strain, no Cfx treatment, mock sample, replicate 2; Escherichia coli; OTHER
For Input DNA (mock samples, treated and non-treated with Cfx) 100 µl of the lysates were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using GeneJET DNA purification kit (Thermo Scientific). DNA fragmentation range was assessed by electrophoresis. For cleavage complexes immunoprecipitation (IP samples, treated and non-treated with Cfx), 900 µl of the remaining lysates were incubated with 100 µL of the ANTI-FLAG M2 affinity gel (Sigma-Aldrich). After immunoprecipitation, the resin was treated with Proteinase K (Sigma-Aldrich) in 200 µL of TES and DNA fragments were purified with AMPure XP magnetic beads from the supernatant (Beckman Coulter). NGS libraries were prepared using Accel NGS 1S kit (Swift Bioscience). DNA sequencing was performed with Illumina HiSeq 4000 in a 150+150 bp paired-end mode. Libraries preparation and sequencing were performed at Skoltech Sequencing Core Facilities. Topo-Seq experiments were performed in triplicate.