Optimized Repli-seq: Improved DNA Replication Timing Analysis by Next-Generation Sequencing

Abstract

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type-specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell analysis of RT has been developed as an alternative, these methods are low throughput and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

Epigenomic signatures associated with spontaneous and replication stress-induced DNA double strand breaks

“…Fixed cells were then sorted by flow cytometry into early and late-replicating fractions. BrdU-labeled DNA from each fraction was immunoprecipitated, followed by preparation for sequencing libraries as previously described (Rivera-Mulia et al, 2022). Two biological replicates were produced for each sample, with similar results.…”

“…These observations are consistent with the known characteristics of the CFSs. To test if the same DSB-associated late-replicating regions remain late-replicating in DMSO or APH treatment, we performed Repli-seq experiments (Rivera-Mulia et al, 2022) with cells treated with DMSO, 0.3 μM or 0.6 μM APH, or nothing at all. Cells were transiently (2 h) labeled by BrdU, followed by sorting into early and late-replicating fractions by flow cytometry.…”