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

## TL;DR

TrAEL-seq is improved to allow high-throughput analysis of DNA replication in mammalian cells without drugs or nucleotides.

## Contribution

The updated TrAEL-seq method enables multiplexing of up to six samples and provides high-resolution replication profiles.

## Key findings

- TrAEL-seq can be used in unsynchronized mammalian cells without nucleotide analogues.
- Replication fork speed is influenced by transcription and proximity to initiation zones.
- Forks accelerate after the first 1 Mb of replication, regardless of transcriptional activity.

## Abstract

TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronized cells and does not require drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq that improves sample quality and includes multiplexing of up to six samples which dramatically improves throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, and instead replication forks accelerate across the first ∼1 Mb of travel irrespective of local transcriptional activity. We propose that this is a consequence of fewer replication forks being active later in S-phase when these distal regions replicate and there being less competition for replication factors.

Graphical Abstract