Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and
late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position.
Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our
Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation
sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled
with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from
both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early
versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide
sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses,
for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct
comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and
molecular biology skills, as well as a basic understanding of Unix and R. |
