ExperimentType 2-stage Repli-seq

DNA replication occurs during the S phase of the cell cycle. In human cells, this process typically lasts for 8–10 h. Different regions of the genome replicate at different times during S phase, following a defined RT program. E/L Repli-seq generates data enabling the study of RT at the genome-wide level, in a sequence-specific manner. The procedure starts with cultured cells that are pulse-labeled with BrdU to label nascent DNA. Cells are then fixed and sorted by flow cytometry on the basis of their DNA content. DNA from early S-phase and late S-phase cells is purified and fragmented. Library construction and BrdU IP are performed in parallel; adaptors are ligated to the purified DNA; next, the BrdU-labeled DNA is immunoprecipitated; and finally, immunoprecipitated DNA is indexed. Indexed and pooled libraries are subsequently sequenced. Analysis of the sequencing data starts with quality control, mapping of reads to the reference genome and calculation of the base 2 log ratio of normalized coverage from the early S fraction to the normalized coverage from the late S fraction in each window (log2 ratio E/L). Data sets to be compared are then quantile-normalized and Loess-smoothed, generating a bedGraph file, which can be used for direct visualization in a genome browser (e.g. http://www.replicationdomain.org) or for further analysis.