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Whole-animal multiplexed single-cell RNA-seq reveals transcriptional shifts across Clytia medusa cell types

Chari, Tara and Weissbourd, Brandon and Gehring, Jase and Ferraioli, Anna and Leclère, Lucas and Herl, Makenna and Gao, Fan and Chevalier, Sandra and Copley, Richard R. and Houliston, Evelyn and Anderson, David J. and Pachter, Lior (2021) Whole-animal multiplexed single-cell RNA-seq reveals transcriptional shifts across Clytia medusa cell types. Science Advances, 7 (48). Art. No. eabh1683. ISSN 2375-2548. PMCID PMC8626072. doi:10.1126/sciadv.abh1683.

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We present an organism-wide, transcriptomic cell atlas of the hydrozoan medusa Clytia hemisphaerica and describe how its component cell types respond to perturbation. Using multiplexed single-cell RNA sequencing, in which individual animals were indexed and pooled from control and perturbation conditions into a single sequencing run, we avoid artifacts from batch effects and are able to discern shifts in cell state in response to organismal perturbations. This work serves as a foundation for future studies of development, function, and regeneration in a genetically tractable jellyfish species. Moreover, we introduce a powerful workflow for high-resolution, whole-animal, multiplexed single-cell genomics that is readily adaptable to other traditional or nontraditional model organisms.

Item Type:Article
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URLURL TypeDescription CentralArticle ItemAll raw sequencing and processed data files Itemsequencing read alignments ItemGoogle Colab notebooks ItemGoogle Colab notebooks expression visualization Paper
Chari, Tara0000-0002-6953-4313
Weissbourd, Brandon0000-0001-5422-3873
Gehring, Jase0000-0002-3894-9495
Ferraioli, Anna0000-0003-1817-6891
Leclère, Lucas0000-0002-7440-0467
Herl, Makenna0000-0001-8518-5179
Gao, Fan0000-0001-6832-3402
Chevalier, Sandra0000-0002-2717-6925
Copley, Richard R.0000-0001-7846-4954
Houliston, Evelyn0000-0001-9264-2585
Anderson, David J.0000-0001-6175-3872
Pachter, Lior0000-0002-9164-6231
Alternate Title:Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types
Additional Information:© 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). Received: 20 February 2021. Accepted: 6 October 2021. We thank X. Da and X. Wang for technical assistance, T. Momose for assistance with the single-cell experimentation, the Caltech Single-Cell Profiling and Engineering Center for the use of their single-cell and sequencing tools, and the Caltech Bioinformatics Resource Center for transcriptome assembly and annotation analysis. We additionally thank the Caltech Center for Evolutionary Science for the bioinformatics resources to create a local UCSC Genome Browser. We thank A. S. Booeshaghi for help with kallisto, bustools, and the kITE demultiplexing of the ClickTag reads and for rescuing the stimulation experiment sequencing data. We thank J. Malamy for helping to establish Clytia work at Caltech. We thank S. Peron for initial characterization of some of the cell type marker genes, P. Lapébie for identification of novel neuropeptide sequences, M. Jager for valuable advice on the in situ protocol, and J. R. Mateu for providing pp11 probe. J.G., M.H., and L.P. were supported in part by a seed grant from the Chen Institute at the California Institute of Technology. T.C., J.G., and L.P. were supported in part by NIH U19MH114830 and NIH RF1AG062324A. We thank the Marine Resources Centre (CRBM and PIV imaging platform) of Institut de la Mer de Villefranche (IMEV), supported by EMBRC-France. The French state funds of EMBRC-France are managed by the ANR within the investments of the Future program. L.L. was supported by the Agence Nationale de la Recherche (ANR-19-CE13-0003). A.F., R.R.C., and E.H. were supported by the H2020/Marie Skłodowska-Curie ITN “EvoCell” Grant agreement no. 766053. B.W. was supported in part by a Howard Hughes Medical Institute Fellowship of the Life Sciences Research Foundation and by NIH K99NS119749. This work was in part supported by the Whitman Center of the Marine Biological Laboratory in Woods Hole, MA and a visiting grant from EMBRC-France. D.J.A. is an Investigator of the Howard Hughes Medical Institute. Author contributions: Conceived of the experiments: T.C., B.W., J.G., R.R.C., E.H., D.J.A., and L.P. Developed cell dissociation, fixation, and labeling procedures compatible with the 10X Genomics platform: J.G. and M.H. Performed the single-cell experiments: T.C., B.W., and J.G. Performed the in situ hybridization and other microscopy experiments: B.W., A.F., L.L., and S.C. Performed whole-organism qPCR: T.C. Performed bioinformatics analysis including assembly and annotation of the transcriptome: F.G. and R.R.C. Wrote scripts for processing the data and code for the analysis: T.C. and J.G. Developed the Google Colab notebooks: T.C. Analyzed and interpreted the data: T.C., B.W., J.G., A.F., L.L., R.R.C., E.H., D.J.A., and L.P. Writing and editing the manuscript: T.C., B.W., J.G., A.F., L.L., R.R.C., E.H., D.J.A., and L.P. The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. All raw sequencing and processed data files used for analysis are available from CaltechData, (, with links additionally provided via the notebooks in the code repository. The sequencing read alignments are available at, and an interactive browser for gene expression visualization (, is publicly hosted on a UCSC Genome Browser by the Caltech Bioinformatics Resource Center. The softwares used are as follows: Cell Ranger 3.0.1, Trinity-v2.8.4, Cufflinks v2.2.1, kallisto v0.46.2, bustools v0.40.0, anndata 0.7.5, louvain 0.7.0, rpy2 3.4.2, scanpy 1.6.0, biopython 1.78, pysam, fuzzywuzzy 0.18.0, numpy 0.19.5, pandas 1.1.5, matplotlib 3.2.2, sklearn 0.0, scipy 1.4.1, seaborn 0.11.1, requests 2.23.0, tqdm 4.41.1, multiprocess, DESeq2 1.3.0, topGO 2.42.0, and UpSet 1.4.0. Code availability: All the codes used to perform the analyses and generate the results and figures are available in Google Colab notebooks archived with Zenodo at and directly available at The notebooks, which include the complete preprocessing of the raw data and a walkthrough of the code, provide a transparent implementation of the methods and can be run for free in the Google cloud.
Group:Tianqiao and Chrissy Chen Institute for Neuroscience
Funding AgencyGrant Number
Caltech Center for Evolutionary ScienceUNSPECIFIED
Tianqiao and Chrissy Chen Institute for NeuroscienceUNSPECIFIED
European Marine Biological Resource CentreUNSPECIFIED
Agence Nationale pour la Recherche (ANR)ANR-19-CE13-0003
Marie Curie Fellowship766053
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Life Sciences Research FoundationUNSPECIFIED
Marine Biological LaboratoryUNSPECIFIED
Issue or Number:48
PubMed Central ID:PMC8626072
Record Number:CaltechAUTHORS:20210126-133110736
Persistent URL:
Official Citation:Chari T, Weissbourd B, Gehring J, Ferraioli A, Leclère L, Herl M, Gao F, Chevalier S, Copley RR, Houliston E, Anderson DJ, Pachter L. Whole-animal multiplexed single-cell RNA-seq reveals transcriptional shifts across Clytia medusa cell types. Sci Adv. 2021 Nov 26;7(48):eabh1683. doi: 10.1126/sciadv.abh1683. Epub 2021 Nov 26. PMID: 34826233.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:107730
Deposited By: Tony Diaz
Deposited On:26 Jan 2021 21:51
Last Modified:07 Dec 2021 21:46

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