Using peCHYRON to Record Mammalian Cells' History of Chemical Exposure into Their DNA

Vertebrate development involves progressive differentiation guided by intrinsic and extrinsic cues; at the population level this yields reproducible outcomes even though single-cell lineage choices can vary between individuals. Variability between individual animals, along with opaque bodies and large numbers of cells, complicates the study of development in vertebrate models. DNA recording technology allows developmental biologists to follow each cell's history, without needing to average across multiple organisms, by stably recording lineage and historical cell signaling as small changes to each cell's genome, which can be read out by DNA sequencing at the end of the experiment [1,2]. Recently, we developed a continuous DNA recording platform, peCHYRON, which allows long-term, multiplexed recording of events in mammalian cells [3]. Here, we describe the design, implementation, and analysis of peCHYRON experiments, demonstrating an example experiment in which constitutive, doxycycline-inducible, and IPTG-inducible gene expression events are recorded concurrently into the DNA of populations of cells. This protocol covers the following steps: the molecular cloning of peCHYRON component plasmids, construction of peCHYRON cell lines, execution of peCHYRON experiments, long-read amplicon sequencing of DNA records, and analysis.