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Lung, spleen and oesophagus tissue remains stable for scRNAseq in cold preservation

E. Madissoon, A. Wilbrey-Clark, R.J. Miragaia, K. Saeb-Parsy, View ORCID ProfileK. Mahbubani, N. Georgakopoulos, P. Harding, K. Polanski, K. Nowicki-Osuch, R.C. Fitzgerald, K.W. Loudon, View ORCID ProfileJ.R. Ferdinand, M.R Clatworthy, A. Tsingene, S. Van Dongen, M. Dabrowska, M. Patel, M.J.T. Stubbington, S. Teichmann, O. Stegle, View ORCID ProfileK.B. Meyer
doi: https://doi.org/10.1101/741405
E. Madissoon
1Wellcome Sanger Institute, Cambridge, UK
2The European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD
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A. Wilbrey-Clark
1Wellcome Sanger Institute, Cambridge, UK
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R.J. Miragaia
1Wellcome Sanger Institute, Cambridge, UK
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K. Saeb-Parsy
3Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, CB2 0QQ
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K. Mahbubani
3Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, CB2 0QQ
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  • ORCID record for K. Mahbubani
N. Georgakopoulos
3Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, CB2 0QQ
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P. Harding
1Wellcome Sanger Institute, Cambridge, UK
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K. Polanski
1Wellcome Sanger Institute, Cambridge, UK
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K. Nowicki-Osuch
4MRC Cancer Unit, Hutchinson-MRC Research Centre, University of Cambridge, Cambridge, CB2 0XZ
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R.C. Fitzgerald
4MRC Cancer Unit, Hutchinson-MRC Research Centre, University of Cambridge, Cambridge, CB2 0XZ
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K.W. Loudon
5Molecular Immunology Unit, Department of Medicine, Cambridge, CB2 0QQ
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J.R. Ferdinand
5Molecular Immunology Unit, Department of Medicine, Cambridge, CB2 0QQ
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  • ORCID record for J.R. Ferdinand
M.R Clatworthy
5Molecular Immunology Unit, Department of Medicine, Cambridge, CB2 0QQ
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A. Tsingene
1Wellcome Sanger Institute, Cambridge, UK
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S. Van Dongen
1Wellcome Sanger Institute, Cambridge, UK
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M. Dabrowska
1Wellcome Sanger Institute, Cambridge, UK
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M. Patel
1Wellcome Sanger Institute, Cambridge, UK
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M.J.T. Stubbington
1Wellcome Sanger Institute, Cambridge, UK
610x Genomics Inc., 6230 Stoneridge Mall Road, Pleasanton, CA 94588, USA
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S. Teichmann
1Wellcome Sanger Institute, Cambridge, UK
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O. Stegle
2The European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD
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K.B. Meyer
1Wellcome Sanger Institute, Cambridge, UK
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  • ORCID record for K.B. Meyer
  • For correspondence: km16@sanger.ac.uk
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Abstract

Background The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single cell RNA sequencing can generate high quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. Despite this, there has not yet been a systematic assessment of the effect of cold storage time on the quality of scRNAseq

Results This study assessed the effect of cold storage on fresh healthy spleen, oesophagus and lung from ≥5 donors over 72 hours. We collected 240,000 high quality single cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these three organs and will allow cross-organ comparison of cell types.

We see little effect of cold ischaemic time on cell viability, yield, total number of reads per cell and other quality control metrics in any of the tissues within the first 24 hours. However, we observed higher percentage of mitochondrial reads, indicative of cellular stress, and increased contamination by background “ambient RNA” reads in the 72h samples in spleen, which is cell type specific.

Conclusions In conclusion, we present robust protocols for tissue preservation for up to 24 hours prior to scRNAseq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

Footnotes

  • em17{at}sanger.ac.uk, aw24{at}sanger.ac.uk, rjudicemiragaia{at}gmail.com, ks10014{at}cam.ac.uk, ktam2{at}cam.ac.uk, ng395{at}cam.ac.uk, pippa.m.harding{at}gmail.com, kp9{at}sanger.ac.uk, kpn25{at}mrc-cu.cam.ac.uk, rcf29{at}mrc-cu.cam.ac.uk, kevinloudon{at}doctors.org.uk, jrf58{at}cam.ac.uk, mrc38{at}medschl.cam.ac.uk. anthitsingene{at}gmail.com, svd{at}sanger.ac.uk, md18{at}sanger.ac.uk, mp20{at}sanger.ac.uk, mjt.stubbington{at}gmail.com, st9{at}sanger.ac.uk, stegle{at}ebi.ac.uk

  • https://prod.data.humancellatlas.org/explore/projects/c4077b3c-5c98-4d26-a614-246d12c2e5d7

  • https://www.tissuestabilitycellatlas.org

  • Abbreviations

    eQTL
    Expression quantitative trait loci
    scRNAseq
    Single cell RNA sequencing
    HCA
    Human Cell Atlas
    DMSO
    Dimethyl sulfoxide
    GTEx
    Genotype-Tissue Expression
    CBTM
    Cambridge Biorepository for Translational Medicine
    DCD
    Donation after cardiac death
    DBD
    Donation after brainstem death
    UW
    University of Wisconsin
    NRP
    Normothermic regional perfusion
    PCR
    Polymerase chain reaction
    RNA
    Ribonucleic acid
    DNA
    Deoxyribonucleic acid
    PBS
    Phosphate buffered saline
    BSA
    Bovine serum albumin
    MACS
    Magnetic activated cell sorting
    FBS
    Foetal bovine serum
    HTA
    Human Tissue Authority
    REC
    Research Ethics Committee
    PMI
    Post mortem interval
    WGS
    Whole Genome Sequencing
    Sc-pseudo-bulk
    Single-cell pseudo-bulk
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Posted August 31, 2019.
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    Lung, spleen and oesophagus tissue remains stable for scRNAseq in cold preservation
    E. Madissoon, A. Wilbrey-Clark, R.J. Miragaia, K. Saeb-Parsy, K. Mahbubani, N. Georgakopoulos, P. Harding, K. Polanski, K. Nowicki-Osuch, R.C. Fitzgerald, K.W. Loudon, J.R. Ferdinand, M.R Clatworthy, A. Tsingene, S. Van Dongen, M. Dabrowska, M. Patel, M.J.T. Stubbington, S. Teichmann, O. Stegle, K.B. Meyer
    bioRxiv 741405; doi: https://doi.org/10.1101/741405
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    Lung, spleen and oesophagus tissue remains stable for scRNAseq in cold preservation
    E. Madissoon, A. Wilbrey-Clark, R.J. Miragaia, K. Saeb-Parsy, K. Mahbubani, N. Georgakopoulos, P. Harding, K. Polanski, K. Nowicki-Osuch, R.C. Fitzgerald, K.W. Loudon, J.R. Ferdinand, M.R Clatworthy, A. Tsingene, S. Van Dongen, M. Dabrowska, M. Patel, M.J.T. Stubbington, S. Teichmann, O. Stegle, K.B. Meyer
    bioRxiv 741405; doi: https://doi.org/10.1101/741405

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