alias: E-MTAB-8946:FX226_p
title: NextSeq 500 paired end sequencing; High-throughput sequencing analysis of the relative frequency of Contacts between Sister Copies emerging from a replication fork in Vibrio cholerae
design: High-throughput sequencing analysis of the relative frequency of Contacts between Sister Copies emerging from a replication fork in Vibrio cholerae
library_construction_protocol: The Cre- and Xer-based SC2 reporters and the Cre-activity reporter were randomly inserted by transposition. Aliquots of the transposon library were thawed on ice and ~a billion cells were diluted into 100 ml of media supplemented with IPTG. Cells were grown until exponential phase (OD600 0.2). Then, ~a billion cells were centrifuged and transferred into 10 ml of fresh media without IPTG. After 30 minutes, arabinose was added at a final concentration of 0.02%. ~109 cells were recovered and frozen in liquid nitrogen at different time points after induction. For the replication arrest assay, rifampicin (300 µg/ml) was added 10 minutes after the induction of the expression of the recombinase.   Extraction of chromosomal DNA was performed using the Sigma GenElute bacterial genomic DNA kit.  Digested DNA was purified with MiniElute columns (Qiagen). The Pippin Prep (SAGE science) was used to purify digested fragments of a length comprised between 800 bp and 2 kbp, which corresponds to the region containing the transposon. 400 ng of purified DNA was ligated overnight at 16 °C to adapters with T4 DNA ligase (NEB). Ligated DNA was purified using MiniElute columns (Qiagen). For each library, 3 PCR reactions were performed using 2 μl of ligated DNA as template and a pair of Illumina oligos. The PCR reactions were performed using the following parameters: 98 °C for 30 seconds, 17 cycles of 98 °C 30 seconds, 68 °C 30 seconds, 72 °C 30 seconds and a final step of 72 °C for 7 minutes. PCR products were concentrated with a MiniElute columns (Qiagen) and purified using Pippin Prep (SAGE science).
identifiers.namespace: Universite Paris-Saclay, CEA, CNRS
identifiers.id: E-MTAB-8946:FX226_p