Table 1.

A summary of the techniques discussed in this review, which were developed to find ways to explore topo binding and cleavage activity genome wide.

Method	Protein	NGS Platform	Brief Description
Type I/II topo ChIP-seq	Type I/II topos	Non-specific	To determine the location and sequence context of topoisomerase binding to DNA using ChIP-seq. Crosslinking agent is applied to cells, before lysis, sonication, and immunoprecipitation of DNA bound to protein of interest. Crosslinking is reversed and DNA is ligated to adapters and sequenced [8].
BLESS	Non-specific	Roche 454 or Illumina Hiseq	To map the location of double strand breaks over the genome using direct in situ Breaks Labelling, Enrichment on Streptavidin and next-generation Sequencing (BLESS). Using human and mouse cells, a fixing agent is used before the cells are lysed and purified nuclei extracted. Biotinylated adapters are used to label DSBs in situ before the DNA is extracted, sonicated and enriched using streptavidin. After a second adapter ligation phase, the DNA is sequenced [9].
END-seq	Non-specific	Illumina Hiseq2500 or Illumina Nextseq500	A more sensitive and robust approach to map DSBs. Mouse lymphocytes or thymocytes are fixed in agarose before being treated with a proteinase K solution, followed by an RNase solution. The biotinylated adapters are then ligated before the DNA is extracted, sonicated and enriched using streptavidin. The DNA then undergoes a second adapter ligation before being sequenced [10]. This protocol was also adapted for use in human and mouse cells looking at topo II cleavage complexes [11].
Spo11-oligo-seq	Spo11	Roche 454	To map the location and sequence context of Spo11 mediated DNA cleavage. Nuclei from meiotic Saccharomyces cerevisiae cultures is extracted, sonicated and ssDNA oligos bound to Spo11 are immunoprecipitated. Proteinase K removes Spo11and DNA is extended at the 3′ end by TdT before a dsDNA adapter is ligated, and the complementary strand synthesized. Adapter 1 contains an inverted dT so the 3′ end is unmodifiable, as is the 5′ end due to the phosphotyrosyl adduct. The DNA then undergoes another 3′ extension and adapter ligation which only affects the newly synthesized strand. The dsDNA fragments are then sequenced [12].
SSB-seq and DSB-seq	Topo II	Illumina GA	To map the location and sequence context of single- and double-stranded DNA breaks induced by Topo II. Human colon cancer cells (HCT116) are treated with etoposide and the genomic DNA is extracted. For single-stranded break (SSB)-seq, SSBs are labelled using nick translation [13] involving digoxigenin labelled dUTP so that fragments can be immunoprecipitated using anti-digoxigenin. For single-stranded break (DSB)-seq, the DNA is 3′-end tailed using TdT in the presence of biotinylated dNTPs and is enriched using streptavidin coated beads. The S1-nuclease is used to cleave the 3′-end tailing and then both the SSB- and DSB-seq DNA populations are adapter ligated and sequenced [14].
CC-seq	Topo II	Illumina Miseq or Nextseq	To map the location and sequence context of Topo II cleavage complexes using cleavage complex (CC)-seq. Human or S. cerevisiae cells are treated with etoposide before lysis. Bulk cellular proteins are removed using a phenol-chloroform extraction and the DNA retained in the aqueous fraction is sonicated. Protein bound DNA is enriched using a silica membrane. The first adapter is ligated to the sonicated DNA-end before Topo II is removed using TDP2, followed by the second adapter ligation and sequencing [15].
NorflIP	Topo IV	Illumina GA	To map the location and sequence context of Topo IV cleavage using nofloxacin immunoprecipitation (NorflIP). Escherichia coli (E. coli) carrying C-terminal FLAG fusions of ParE and ParC is treated with norfloxacin before lysis, sonication and immune-precipitation using anti-FLAG. Adapters are ligated and fragments sequenced [16,17].
Topo-seq	DNA gyrase	Illumina Nextseq	To map the location and sequence context of DNA gyrase cleavage. E. coli cells are treated with ciprofloxacin, oxolinic acid or microcin B17, before lysis and sonication. DNA bound to gyrase is enriched using immunoprecipitation and proteinase K is used to remove gyrase. The DNA is then treated using the Accel 1S NGS kit, which ligates adapters to the 3′ strand from the gyrase cleavage site (5′ strand is unmodifiable due to the phosphotyrosyl adduct), and synthesizes the complementary strand. The resultant dsDNA is then sequenced [18].
Top1-seq	Topo 1B	Illumina GAII and SoLid (applied biosystems)	To map the location and sequence context of Topo 1B cleavage. Human colon cancer cells (HCT116) are briefly treated with camptothecin before being lysed, sonicated and immunoprecipitated. Topo 1B is proteolyzed and the DNA ligated to adapters and sequenced [19].