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Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis

Nature Protocols volume 10, pages 974–984 (2015)Cite this article

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Abstract

Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.

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Figure 1: Scheme for single prokaryotic cell isolation by LCM and total transcript amplification.
Figure 2: Process of single-cell isolation using LCM (Step 4).
Figure 3: Anticipated results from Step 27 showing the DNase I random fragmentation of amplified ds-cDNA on an agarose gel.
Figure 4: Statistical analyses of transcriptomic data from multiple B. thailandensis single-cell experiments.

References

  1. Kurimoto, K. et al. An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic Acids Res. 34, e42 (2006).

    Article  Google Scholar 

  2. Scanlon, M.J., Ohtsu, K., Timmermans, M.C. & Schnable, P.S. Laser Microdissection-Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA (John Wiley & Sons, Inc., 2009).

  3. Emmert-Buck, M.R. et al. Laser capture microdissection. Science 274, 998–1001 (1996).

    Article  CAS  Google Scholar 

  4. Tietjen, I. et al. Single-cell transcriptional analysis of neuronal progenitors. Neuron 38, 161–175 (2003).

    Article  CAS  Google Scholar 

  5. Iscove, N.N. et al. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat. Biotechnol. 20, 940–943 (2002).

    Article  CAS  Google Scholar 

  6. Tougan, T., Okuzaki, D. & Nojima, H. Chum-RNA allows preparation of high-quality cDNA library from sing-cell quantity of mRNA without PCR amplification. Nucleic Acids Res. 36, e92 (2008).

    Article  Google Scholar 

  7. Kurimoto, K., Yabuta, Y., Ohinata, Y. & Saitou, M. Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat. Protoc. 2, 739–752 (2007).

    Article  CAS  Google Scholar 

  8. Kang, Y. et al. Transcript amplification from single bacterium for transcriptome analysis. Genome Res. 21, 925–935 (2011).

    Article  CAS  Google Scholar 

  9. Passalacqua, K.D. et al. Structure and complexity of a bacterial transcriptome. J. Bacteriol. 191, 3203–3211 (2009).

    Article  CAS  Google Scholar 

  10. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 2nd edn (Cold Spring Harbor Laboratory Press, 2001).

  11. Son, M.S., Matthews, W.J.J., Kang, Y., Nguyen, D.T. & Hoang, T.T. In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infect. Immun. 75, 5313–5324 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This project was supported by the US National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) grant number R01GM103580 and by the Center of Biomedical Research Excellence grant P20GM103516 from the National Center for Research Resources of the National Institutes of Health, and it was partially supported by Award U54 AI065359 from the National Institute of Allergy and Infectious Diseases. We acknowledge R. Hendrickson for his help with editing part of this protocol.

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  1. Michael H Norris

    Present address: Present address: Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, USA.,

Authors and Affiliations

  1. Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    Yun Kang & Tung T Hoang

  2. Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    Ian McMillan, Michael H Norris & Tung T Hoang

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  1. Yun Kang
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  2. Ian McMillan
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  3. Michael H Norris
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Contributions

T.T.H., Y.K. and M.H.N. designed the experiments. Y.K., I.M. and M.H.N. performed the experiments. Y.K., I.M., M.H.N. and T.T.H. wrote this manuscript.

Corresponding author

Correspondence to Tung T Hoang.

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The authors declare no competing financial interests.

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Kang, Y., McMillan, I., Norris, M. et al. Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis. Nat Protoc 10, 974–984 (2015). https://doi.org/10.1038/nprot.2015.058

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