Topo-Seq application for topoisomerases binding sites identification with a single-nucleotide resolution

Topo-Seq is a ChIP-Seq-based methodology that allows high-throughput identification of topoisomerases binding (cleavage) sites with a single-base precision. On a first stage of the project DNA-gyrase binding sites on a Escherichia coli DY330 genome are investigated. Overall design: Escherichia coli DY330 cultures were treated with a range of DNA-gyrase poisons (ciprofloxacin, microcin B17, oxolinic acid) during exponential phase (OD600=0.6), than cells were harvested and lysed with sonication. Complexes of gyrase with DNA were immunoprecipitated using ANTI-FLAG M2 affinnity resin, proteins were degraded with proteinase K and DNA extracted by phenol-chlorophorm followed by precipitation in alcohol. Lybraries from the DNA recovered were prepared using NGS Accel 1S Plus DNA kit (Swift Bioscience). Reads were mapped to E. coli w3110 MuSGS genome (with BWA MEM), signals (Gyrase Cleavage Sites - GCSs) were identified using Audic-Claveiry statistic (custom scripts with detailed descriptions could be found at https://github.com/sutormin94/Gyrase_Topo-seq).