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.

本研究采用双端深度测序（Paired-end deep-sequencing）技术，平行检测随机整合于细胞文库不同基因组位点的姐妹染色单体接触（SC2）报告因子及/或位点特异性重组酶（SSR）活性报告因子的状态与定位。其中，SC2报告因子为拓扑非依赖型位点特异性重组酶（Cre重组酶或Xer重组酶）适配的短重组盒；SSR活性报告因子则为长重组盒。SC2报告因子盒由两段正向重复的重组位点构成，二者间隔的DNA片段过短，无法通过分子内重组实现位点切除；而SSR活性重组盒同样由两段正向重复的重组位点构成，但其间隔的DNA片段长度足以通过分子内重组实现位点切除。实验首先构建携带Cre重组酶或Xer重组酶条件性表达等位基因的细胞系，并创建在不同基因组位置整合有同源SC2或SSR活性报告因子的细胞文库。随后在细胞生长的不同时长时段及/或细胞周期的特定阶段诱导重组酶的表达。最终通过高通量双端深度测序，确定每个细胞所携带的SC2报告因子的基因组位置，以及其所含重组盒的重组状态。