scRIPT: a rapid, high-coverage single-cell lineage-tracing method applied to the hematoendothelial differentiation of embryonic stem cells

Cetin, R.; Bindels, E.; van Staalduinen, J.; Kuijpers, L.; Picco, G.; Sanders, M. A.; Hoogenboezem, R. M.; van Beek, G.; Gribnau, J.; Mulugeta, E.; Huylebroeck, D.; Grosveld, F.

doi:10.64898/2026.10.14.759042

bioRxiv · 10.64898/2026.10.14.759042

scRIPT: a rapid, high-coverage single-cell lineage-tracing method applied to the hematoendothelial differentiation of embryonic stem cells

Cetin, R.·Bindels, E.·van Staalduinen, J.·Kuijpers, L.·Picco, G.·Sanders, M. A.·Hoogenboezem, R. M.·van Beek, G.·Gribnau, J.·Mulugeta, E.·Huylebroeck, D.·Grosveld, F.

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Abstract

Single-cell lineage tracing, which often involves CRISPR-Cas-based barcode insertion followed by novel barcode creation, links a living cell's transcriptome to its clonal history. It is often less suitable for short cell differentiation protocols: the barcoding time window does not always align well with that of differentiation, barcode dropout rates can be too high, library preparation remains laborious, and many studies lack appropriate lineage-specific quality control. We developed scRIPT (single-cell Rapid Integrated Platform for Tracing), which combines rapid ribonucleoprotein (RNP)-based barcoding with fluorescent reporter coding sequence insertion, reporter-based enrichment of informative cells by cell sorting, streamlined library preparation, and dual short-read sequencing, coupled to dedicated quality-control metrics for lineage data. scRIPT achieves a low cell dropout rate (<5% of cells presenting without a detectable lineage barcode) and a reliable assignment of barcodes (of >90% of cells). We applied scRIPT to document mouse embryonic stem cell (ESC) differentiation into early hematoendothelial cells and revealed clonal relationships and fate coupling among the emerging cell (sub)types. A subset of cell clones was biased towards single fates, and bifurcating progenitors already express mRNA of canonical regulators of their eventual fate. The results also suggest a heritable, fate-independent clonal memory enriched with imprinted genes. Taken together, scRIPT provides a convenient, rapid, reliable, high-coverage, and quality-controlled platform that combines cell lineage and transcriptome readouts.

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Cetin, R., Bindels, E., van Staalduinen, J., Kuijpers, L., Picco, G., Sanders, M. A., Hoogenboezem, R. M., van Beek, G., Gribnau, J., Mulugeta, E., Huylebroeck, D., Grosveld, F.. 2026-10-16. scRIPT: a rapid, high-coverage single-cell lineage-tracing method applied to the hematoendothelial differentiation of embryonic stem cells. https://doi.org/10.64898/2026.10.14.759042

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