High-throughput sequencing analysis of the relative frequency of Contacts between Sister Copies emerging from a replication fork in Vibrio cholerae. High-throughput sequencing analysis of the relative frequency of Contacts between Sister Copies emerging from a replication fork in Vibrio cholerae

Paired-end deep-sequencing was used to determine in parallel the status and position of sister-chromatid contact (SC2) reporters and/or site-specific recombinase (SSR) activity reporters inserted at random positions in a library of cells. The SC2 reporters are short recombination cassettes for a topology-independent site-specific recombinase (Cre or Xer). The SSR-activity reporters are long recombination cassettes. The SC2 reporter cassettes are composed of two directly-repeated recombination sites separated by a DNA segment too short to permit their excision by intramolecular recombination. The SSR-activity cassettes are composed of two directly-repeated recombination sites separated by a DNA segment long enough to permit their excision by intramolecular recombination. The assays start with the engineering of a cell line with a conditional expression allele for Cre or Xer and the creation of a library of cells harbouring a cognate SC2 or SSR-activity reporter at different genomic positions. Production of the recombinase was induced for different lengths of time during cell growth and/or at specific stages of the cell cycle. The position of the SC2 reporter harboured by each cell and the recombination status of the recombination cassette it contains are then determined by high-throughput paired-end sequencing.