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Method Article
Alexander van Oudenaarden
AvO lab, Hubrecht Institute
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
ScarTrace is a protocol that allows the simultaneous amplification of the transcriptome and Cas9-induced genetic scars of thousands of zebrafish single cells. Initially, Tg(H2Af/va-GFP)(kca66) zebrafish one-cell embryos are injected with Cas9 RNA or protein and a gRNA targeting a GFP transgene to allow scaring. At adulthood, various zebrafish organs are dissected, dissociated and single cells are sorted. The following protocol describes the steps to generate transcriptome and scar sequencing libraries from single cells from the WKM, brain, eyes and caudal fin. Briefly, ScarTrace is an adaptation of the SORT-Seq protocol1 where a nested PCR step was integrated to amplify scars after mRNA conversion to cDNA.
Keywords
single-cell, transcriptome, Cas9-induced genetic scar, clone-tracing
The authors declare no competing financial interests.
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Method Article
Alexander van Oudenaarden
AvO lab, Hubrecht Institute
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 12 Jun, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
ScarTrace is a protocol that allows the simultaneous amplification of the transcriptome and Cas9-induced genetic scars of thousands of zebrafish single cells. Initially, Tg(H2Af/va-GFP)(kca66) zebrafish one-cell embryos are injected with Cas9 RNA or protein and a gRNA targeting a GFP transgene to allow scaring. At adulthood, various zebrafish organs are dissected, dissociated and single cells are sorted. The following protocol describes the steps to generate transcriptome and scar sequencing libraries from single cells from the WKM, brain, eyes and caudal fin. Briefly, ScarTrace is an adaptation of the SORT-Seq protocol1 where a nested PCR step was integrated to amplify scars after mRNA conversion to cDNA.
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