Repli-seq maps genome-wide replication timing by sequencing newly synthesized DNA from cells isolated at different stages of S phase. By measuring the relative replication activity of genomic regions throughout S phase, Repli-seq defines early-, intermediate-, and late-replicating domains. Replication timing is closely associated with genome organization and chromatin states: early-replicating regions are generally enriched in gene-rich, transcriptionally active euchromatin, whereas late-replicating regions are often associated with gene-poor, transcriptionally repressed heterochromatin. Therefore, Repli-seq provides an important functional readout of genome architecture and chromatin regulation.