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

bioRxiv (Cold Spring Harbor Laboratory)

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.

Authors 12

1. Rıdvan Çeti̇n

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

2. Eric M. Bindels

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

3. Jente van Staalduinen

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

4. Lucas Kuijpers

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

5. Giulia Picco

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

6. Mathijs Arnoud Sanders

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

7. Remco Michiel Hoogenboezem

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

8. Gregory van Beek

   Erasmus MC

   Affiliation as printed

   Erasmus MC;

9. Joost H. Gribnau Aachen

   Leiden University Medical Center

   Affiliation as printed

   LUMC

10. Eskeatnaf Mulugeta

    Erasmus MC

    Affiliation as printed

    Erasmus MC;

11. Danny Huylebroeck

    Erasmus MC

    Affiliation as printed

    Erasmus MC;

12. Frank G. Grosveld

    Erasmus MC

    Affiliation as printed

    Erasmus MC;