Single pulse experiments | S-phase progression
In a human cell thousands of replication forks simultaneously coordinate the duplication of the entire genome. The rate at which this process occurs might depend on the epigenetic state of the genome and vary between, or even within, cell types. To accurately measure DNA replication speeds, we developed a technology to detect recently replicated DNA using single-cell sequencing. We observed that replication speed is not constant but increases during S-phase of the cell cycle. Using genetic and pharmacological perturbations we are able to alter this acceleration of replication and conclude that DNA damage inflicted by the process of transcription limits the speed of replication during early S-phase. In late S-phase, during which less transcribed regions replicate, replication accelerates and approaches its maximum speed.
scEdU-seq relies on metabolic labeling with the nucleotide analog 5-Ethynyl-2′-deoxyuridine (EdU) and subsequent affinity capture of newly synthesized DNA fragments. We make use of CuAAC Click chemistry to covalently link a biotin moiety to the uracil base. Subsequently, we digest the single-cell genome and ligate adapters containing a T7 promoter, cell-specific barcodes and a unique molecular identifier (UMI). After pooling cells, we biotin-capture the EdU containing DNA molecules and release the non-EdU modified strand by heat denaturation. Finally, we regenerate the complementary strand via primer extension followed by amplification to prepare for sequencing.
Jeroen van den Berg, Vincent van Batenburg, Christoph Geisenberger, Rinskje B. Tjeerdsma, Anchel de Jaime-Soguero, Sergio P. Acebrón, Marcel A. T. M. van Vugt and Alexander van Oudenaarden. Nature Methods (2024): 2024-06.
link to the paperJan Dreyer, Giulia Ricci, Jeroen van den Berg, Vivek Bhardwaj, Janina Funk, Claire Armstrong, Vincent van Batenburg, Chance Sine, Michael A. VanInsberghe, Richard Marsman, Imke K. Mandemaker, Simone di Sanzo, Juliette Costantini, Stefano G. Manzo, Alva Biran, Claire Burny, Moritz Völker-Albert, Anja Groth, Sabrina L. Spencer, Alexander van Oudenaarden, Francesca Mattiroli. bioRxiv (2024): 2024-03.