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transgenic killifish, 15–20 pg DNA was co-injected with 30 pg transposase mRNA
into one-cell-stage N. furzeri embryos and the injected embryos were maintained
in Yamamoto embryo solution (17 mM NaCl, 2.7 mM KCl, 2.5 mM CaCl 2 , 0.02
mM NaHCO 3 , pH 7.3) at 28 °C for 2 weeks before hatching. F 0 founders were
crossed with wild-type GRZ fish and three independent lines were established
for gene expression studies. No genotyping was performed to detect the trans-
gene. However, for the first transgenic line, 15 out of 46 F 1 embryos showed GFP
expression in the forebrain. For the second transgenic line, 8 out of 25 F 1 embryos
showed GFP expression in the forebrain. Finally, 10 out 37 F 1 embryos of the third
transgenic line had GFP expression in the forebrain. No particular randomization
strategy was implemented.
Killifish embryos were removed manually from the chorion before imaging.
The juvenile fish were anaesthetized in 150 mg/l MS-222 for 5 min at room tem-
perature. Images were taken with Ultraview R2 spinning disk confocal microscope.
Estimation of sample size, blinding and randomization. No statistical methods
were used to predetermine sample size. For the single-cell experiments, because
the embryo collection and the subsequent data analysis was performed by different
researchers, the investigators were blinded to group allocation. For the functional
assays, no particular blinding strategy was adopted. As stated in the specific sec-
tions above, the assignment of the Ciona embryos to the different conditions were
randomized. Otherwise, no particular randomization strategy was used.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this paper.Data availability
Raw sequencing data and the gene-expression matrix are available in the Gene
Expression Omnibus (GEO) under accession number GSE131155. Our data can
be explored at https://portals.broadinstitute.org/single_cell/study/SCP454/com-
prehensive-single-cell-transcriptome-lineages-of-a-proto-vertebrate. All other data
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Acknowledgements We thank J. B. Wiggins, J. M. Miller and the Genomics Core
Facility for technical support of 10x Chromium platform; IT and Bioinformatics
staff at the Lewis-Sigler Institute for Integrative Genomics (LSI) for development
of the sequence alignment pipeline; E. G. Gatzogiannis (director of the LSI
Imaging Core Facility) for building the two-photon microscope and help with
imaging; the Shvartsman laboratory in LSI for Imaris access; A. Sánchez
Alvarado at SIMR for providing laboratory support and resources; and members
of the Levine Laboratory for helpful discussions, and especially N. Treen for
suggesting mNeonGreen as fluorescent reporter. This study was supported by a
grant from the NIH (NS076542) to M.L. W.W. (SIMR) was funded by the Stowers
Institute.Author contributions K.C. and M.L. conceived the project. K.C., M.L., C.C.,
L.A.L. and W.W. (SIMR) designed the experiments. L.A.L., P.H.Y., Y.A.C. and K.C.
performed Ciona experiments, W.W. (SIMR) performed killifish experiments, C.C.
performed computational data analysis. L.R.P., J.C.M. and W.W. (LSI) set up the
single-cell RNA-sequencing pipeline. M.L. supervised the project. All authors
contributed to interpretation of the results, and C.C., L.A.L., K.C. and M.L. wrote
the manuscript.Competing interests The authors declare no competing interests.Additional information
supplementary information is available for this paper at https://doi.org/
10.1038/s41586-019-1385-y.
Correspondence and requests for materials should be addressed to M.L. or K.C.
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