Abstract
Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR–Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods. The recorded lineages are captured, along with thousands of cellular transcriptomes, to build lineage trees with hundreds of branches representing relationships among profiled cell types. Here, we provide details for (i) generating transgenic zebrafish; (ii) performing multi-timepoint barcode editing; (iii) building scRNA-seq libraries from brain tissue; and (iv) concurrently amplifying lineage barcodes from captured single cells. Generating transgenic lines takes 6 months, and performing barcode editing and generating single-cell libraries involve 7 d of hands-on time. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration, or disease.
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Data availability
Figure 4 has associated raw data (Supplementary Data). There is no restriction on data availability. scGESTALT computational scripts and analysis pipeline are available at https://github.com/aaronmck/SC_GESTALT and are included as Supplementary Software 2 with this protocol.
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Acknowledgements
We thank D.E. Wagner, A. McKenna, and S. Pandey for discussion and advice. This work was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research to B.R.; NIH grants U01MH109560, R01HD85905, and DP1 HD094764 to A.F.S.; and an Allen Discovery Center grant to A.F.S.
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B.R. and J.A.G. developed and optimized the scGESTALT protocols and analyzed the data. B.R. wrote the manuscript with edits by A.F.S. and J.A.G.
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Key references using this protocol
Raj, B. et al. Nat. Biotechnol. 36, 442–450 (2018): https://doi.org/10.1038/nbt.4103
McKenna, A. et al. Science 353, aaf7907 (2016): https://doi.org/10.1126/science.aaf7907
Integrated supplementary information
Supplementary Figure 1 Papain oxygenation setup.
Left and middle panels, 95%O2: 5%CO2 gas tank fitted with a gas regulator, Tygon E-3603 tubing and a 5 ml serological pipette. Right panel, Oxygenation of papain/DNase mix in Neurobasal Medium (small vial to the right) is performed by bubbling 95%O2: 5%CO2 gas through tubing attached to a sterile 5 ml serological pipette for 2 min (Procedure Step 52). EBSS buffer (large vial to the left, used for resuspending ovumucoid, Procedure Step 53) and Neurobasal Medium (Procedure Step 50) are oxygenated in a similar manner.
Supplementary Figure 2 Zebrafish brain dissection.
Top panels, Anesthetized fish is transferred to a Sylgard dish covered with Neurobasal Medium and MESAB (left). The fish is pinned just posterior of the head, in the middle of the trunk and near the tail using 3 insect pins (right, asterisks mark pin positions). Bottom panels, The jaw, eyes, heart and gut tissues are removed. The skin on top of the head is pierced and peeled back to expose the brain (left, circle marks the exposed brain). Gently scoop the brain out taking care not to lose part of the hindbrain in the process (right, whole brain is encircled).
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Supplementary Figures 1 and 2, Supplementary Tables 1 and 2
Supplementary Video 1
Papain–DNase mix oxygenation
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Raj, B., Gagnon, J.A. & Schier, A.F. Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR–Cas9 barcodes by scGESTALT. Nat Protoc 13, 2685–2713 (2018). https://doi.org/10.1038/s41596-018-0058-x
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DOI: https://doi.org/10.1038/s41596-018-0058-x
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