GSE135178
DNA copy number measurement of genome replication dynamics by high-throughput sequencing – the sort-seq, sync-seq and MFA-seq family
Genome replication follows a defined temporal programme that can change during cellular differentiation and disease onset. DNA replication results in an increase in DNA copy number that can be measured by high-throughput sequencing (HTS). Here we present a protocol to determine genome replication dynamics using DNA copy number measurements. Cell populations can be obtained in three variants of the method. First, sort-seq uses fluorescence activated cell sorting (FACS) to isolate replicating and non-replicating subpopulations from asynchronous cells. Second, sync-seq uses cell synchronisation to obtain samples before and during DNA replication. Third, marker frequency analysis (MFA-seq) assesses copy number differences in rapidly replicating asynchronous cells. These approaches have been used to reveal genome replication dynamics in prokaryotes, archaea, and a wide range of eukaryotes, including mammalian cells. The whole protocol can be performed in 7-10 days. Overall design: S. cerevisiae strains from mid-log phase were sorted based on DNA content, enriching for S phase cells. Extracted DNA from the cells was sequenced on NextSeq 500 and unique reads were counted in genomic bins of 1 kb. Examples of aneuploidy (RWY012, chrXI and chrXII) and segmental duplication (RWY109, region of chrIV).