A new method, scEdU-seq, is introduced to measure DNA replication speeds in single cells. This method uses 5-ethynyl-2'-deoxyuridine (EdU) labeling and single-cell sequencing to detect nascent DNA. The study shows that DNA replication speed increases during S phase of the cell cycle. In early S phase, replication is slowed by transcription, while in late S phase, replication accelerates. The method allows high-resolution analysis of DNA replication fork dynamics and speeds. Using a double-pulse EdU labeling strategy, the researchers estimate DNA replication speeds by analyzing the distance between EdU-labeled regions. They find that transcription limits DNA replication speeds in early S phase, as transcription-coupled DNA damage activates PARP1, which in turn slows replication. Inhibition of transcription or PARP activity increases replication speeds. The study also shows that DNA replication speeds vary between cells and that factors such as chromatin state and transcriptional activity influence replication speed. scEdU-seq provides a powerful tool for studying DNA replication dynamics in single cells, with applications in understanding replication stress, genomic instability, and the role of transcription in DNA replication. The method is scalable and cost-effective, making it suitable for large-scale studies. The findings highlight the importance of transcription in regulating DNA replication speed and suggest that transcription-coupled DNA damage plays a key role in limiting replication speed during early S phase.A new method, scEdU-seq, is introduced to measure DNA replication speeds in single cells. This method uses 5-ethynyl-2'-deoxyuridine (EdU) labeling and single-cell sequencing to detect nascent DNA. The study shows that DNA replication speed increases during S phase of the cell cycle. In early S phase, replication is slowed by transcription, while in late S phase, replication accelerates. The method allows high-resolution analysis of DNA replication fork dynamics and speeds. Using a double-pulse EdU labeling strategy, the researchers estimate DNA replication speeds by analyzing the distance between EdU-labeled regions. They find that transcription limits DNA replication speeds in early S phase, as transcription-coupled DNA damage activates PARP1, which in turn slows replication. Inhibition of transcription or PARP activity increases replication speeds. The study also shows that DNA replication speeds vary between cells and that factors such as chromatin state and transcriptional activity influence replication speed. scEdU-seq provides a powerful tool for studying DNA replication dynamics in single cells, with applications in understanding replication stress, genomic instability, and the role of transcription in DNA replication. The method is scalable and cost-effective, making it suitable for large-scale studies. The findings highlight the importance of transcription in regulating DNA replication speed and suggest that transcription-coupled DNA damage plays a key role in limiting replication speed during early S phase.