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Contemporary Approaches to the Study of Pain pp 17–38Cite as

Preparation of Human and Rodent Spinal Cord Nuclei for Single-Nucleus Transcriptomic Analysis

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Part of the book series: Neuromethods ((NM,volume 178))

Abstract

The spinal cord is a complex and heterogeneous tissue that is composed of numerous neuronal and non-neuronal cell types. Single-cell RNA-seq has emerged as a powerful method to study heterogeneous tissues by allowing for the capture and analysis of individual cells. A key step in the analysis of solid tissues is the dissociation of the tissue into single cells. While many tissues can be readily dissociated by enzymatic or mechanical methods, highly interconnected tissues such as the spinal cord are especially difficult to dissociate, leading to reductions in cellular viability or large biases in the representation of cell types. Moreover, it has been shown that dissociation of live cells can induce gene expression changes that influence downstream analysis. Single-nucleus RNA-seq (snRNA-seq) offers an alternative method of studying the transcriptomes of individual cells that circumvents many of these issues. An additional benefit of snRNA-seq is that it can be performed on frozen tissues, thus opening the door to the study of biobanked pathological human tissues. We present here a straightforward protocol to isolate both murine and human spinal cord nuclei for transcriptomic analysis on multiple platforms, including 10× Genomics Chromium.

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References

  1. Ji R, Berta T, Nedergaard M (2013) Glia and pain: is chronic pain a gliopathy? Pain 154:S10–S28

    Article  Google Scholar 

  2. Ji R-R, Chamessian A, Zhang Y-Q (2016) Pain regulation by non-neuronal cells and inflammation. Science 354:572–577

    Article  CAS  Google Scholar 

  3. Peirs C, Seal RP (2016) Neural circuits for pain: Recent advances and current views. Science 354:578–584

    Article  CAS  Google Scholar 

  4. Zeisel A, Hochgerner H, Lönnerberg P et al (2018) Molecular architecture of the mouse nervous system. Cell 174:999–1014.e22

    Article  CAS  Google Scholar 

  5. Häring M, Zeisel A, Hochgerner H et al (2018) Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types. Nat Neurosci 61:1

    Google Scholar 

  6. Sathyamurthy A, Johnson KR, Matson KJE et al (2018) Massively parallel single nucleus transcriptional profiling defines spinal cord neurons and their activity during behavior 22:2216–2225

    CAS  Google Scholar 

  7. Rosenberg AB, Roco CM, Muscat RA et al (2018) Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 12:eaam8999

    Google Scholar 

  8. Denk F, Crow M, Didangelos A et al (2016) Persistent alterations in microglial enhancers in a model of chronic pain. Cell Rep 15:1771–1781

    Article  CAS  Google Scholar 

  9. Machado L, Geara P, Camps J et al (2021) Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation. Cell Stem Cell 28(6):1125–1135.e7

    Article  CAS  Google Scholar 

  10. Lacar B, Linker SB, Jaeger BN et al (2016) Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat Commun 7:11022

    Article  CAS  Google Scholar 

  11. Chamessian A, Young M, Qadri Y et al (2018) Transcriptional profiling of somatostatin interneurons in the spinal dorsal horn. Sci Rep 8:6809

    Article  Google Scholar 

  12. Serafin EK, Chamessian A, Li J et al (2019) Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse. Pain 160:2380–2397

    Article  CAS  Google Scholar 

  13. Macosko EZ, Basu A, Satija R et al (2015) Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161:1202–1214

    Article  CAS  Google Scholar 

  14. Hagemann-Jensen M, Ziegenhain C, Chen P et al (2020) Single-cell RNA counting at allele and isoform resolution using smart-seq3. Nat Biotechnol 38(6):708–714

    Article  CAS  Google Scholar 

  15. Slyper M, Porter CBM, Ashenberg O et al (2020) A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat Med 26:792–802

    Article  CAS  Google Scholar 

  16. Schirmer L, Velmeshev D, Holmqvist S et al (2019) Neuronal vulnerability and multilineage diversity in multiple sclerosis. Nature 388:1

    Google Scholar 

  17. Krishnaswami SR, Grindberg RV, Novotny M et al (2016) Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons 11:499–524

    CAS  Google Scholar 

  18. Martelotto L ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue for snRNAseq. https://www.protocols.io/view/frankenstein-protocol-for-nuclei-isolation-from-f-3fkgjkw

  19. Matson KJE, Sathyamurthy A, Johnson KR et al (2018) Isolation of adult spinal cord nuclei for massively parallel single-nucleus RNA sequencing. J Vis Exp (140):e58413

    Google Scholar 

  20. Nott A, Schlachetzki JCM, Fixsen BR et al (2021) Nuclei isolation of multiple brain cell types for omics interrogation. Nat Protoc 16(3):1629–1646

    Article  CAS  Google Scholar 

  21. Grindberg RV, Yee-Greenbaum JL, McConnell MJ et al (2013) RNA-sequencing from single nuclei. Proc Natl Acad Sci U S A 110:19802–19807

    Article  CAS  Google Scholar 

  22. Hodge RD, Bakken TE, Miller JA et al (2019) Conserved cell types with divergent features in human versus mouse cortex. Nature 573:61–68

    Article  CAS  Google Scholar 

  23. Sun W, Cornwell A, Li J et al (2017) SOX9 is an astrocyte-specific nuclear marker in the adult brain outside the neurogenic regions. J Neurosci 37:4493–4507

    Article  CAS  Google Scholar 

  24. Okada S, Saiwai H, Kumamaru H et al (2011) Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue. J Cell Physiol 226:552–558

    Article  CAS  Google Scholar 

  25. Sasagawa Y, Danno H, Takada H et al (2018) Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads. Genome Biol 19:14049

    Article  Google Scholar 

  26. Malin SA, Davis BM, Molliver DC (2007) Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc 2:152–160

    Article  CAS  Google Scholar 

  27. Richner M, Jager SB, Siupka P et al (2017) Hydraulic extrusion of the spinal cord and isolation of dorsal root ganglia in rodents. J Vis Exp (119):55226

    Google Scholar 

  28. Duggal N, Lach B (2002) Selective vulnerability of the lumbosacral spinal cord after cardiac arrest and hypotension. Stroke 33:116–121

    Article  CAS  Google Scholar 

  29. Hagemann-Jensen M, Ziegenhain C, Chen P et al (2019) Single-cell RNA counting at allele- and isoform-resolution using smart-seq3. Biorxiv 817924

    Google Scholar 

  30. Hodge RD, Bakken TE, Miller JA, Smith KA, Barkan ER, Graybuck LT, Close JL, Long B, Johansen N, Penn O et al (2019) Conserved cell types with divergent features in human versus mouse cortex. Nature 536:1–8

    Google Scholar 

  31. Picelli S, Björklund ÅK, Faridani OR et al (2013) Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096–1098

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Anesthesiology and Neurology, Washington University in Saint Louis School of Medicine, Saint Louis, MO, USA

    Alexander Chamessian

  2. Department of Anesthesiology, Pain Research Center, University of Cincinnati Medical Center, Cincinnati, OH, USA

    Temugin Berta

Authors
  1. Alexander Chamessian
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  2. Temugin Berta
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Corresponding author

Correspondence to Temugin Berta .

Editor information

Editors and Affiliations

  1. Department of Neurobiology, Pittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, PA, USA

    Rebecca P. Seal

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Chamessian, A., Berta, T. (2022). Preparation of Human and Rodent Spinal Cord Nuclei for Single-Nucleus Transcriptomic Analysis. In: Seal, R.P. (eds) Contemporary Approaches to the Study of Pain. Neuromethods, vol 178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2039-7_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2039-7_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2038-0

  • Online ISBN: 978-1-0716-2039-7

  • eBook Packages: Springer Protocols

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