Multiplexed TrAEL-seq captures DNA replication dynamics in mammalian cells.

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• DNA replication is a fundamental cellular process essential for cell division and genomic stability.
• Accurate genome-wide profiling of DNA replication is crucial for understanding various biological phenomena, including development, aging, and disease.
• Existing methods for DNA replication profiling often have limitations such as requiring synchronized cells or specific chemical treatments.

Purpose of the Study:

• To present an updated and improved protocol for TrAEL-seq (truncated adapter ligation sequencing).
• To enhance the throughput and sample quality of TrAEL-seq.
• To validate the enhanced TrAEL-seq method in multiple mammalian cell lines and dynamic biological systems.

Main Methods:

• The study details an updated TrAEL-seq protocol.
• The method involves multiplexing up to six samples for increased throughput.
• The protocol was validated in various mammalian cell lines, including during oncogene-induced senescence.

Main Results:

• The updated TrAEL-seq protocol provides high-resolution genome-wide DNA replication profiles comparable to existing methods like OK-seq.
• The method successfully mapped replication initiation and termination zones.
• TrAEL-seq revealed insights into replication fork speed, showing slower progression through transcribed regions but overall acceleration in early S-phase.

Conclusions:

• The enhanced TrAEL-seq method is a robust, high-throughput, and high-resolution technique for profiling DNA replication genome-wide.
• This method is suitable for analyzing replication dynamics in unsynchronized cells and complex biological systems.
• The findings suggest that replication fork progression is influenced by transcriptional activity and proximity to initiation zones, with fewer competing forks later in S-phase.