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Letters to the Editor
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Haojia Wu, Yuhei Kirita, Erinn L. Donnelly and Benjamin D. Humphreys
JASN April 2019, 30 (4) 714; DOI: https://doi.org/10.1681/ASN.2019020178
Haojia Wu
1Division of Nephrology, Department of Medicine and
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Yuhei Kirita
1Division of Nephrology, Department of Medicine and
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Erinn L. Donnelly
1Division of Nephrology, Department of Medicine and
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Benjamin D. Humphreys
1Division of Nephrology, Department of Medicine and
2Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, Missouri
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  • RNA-sequencing
  • Cell dissociation
  • fibrosis

We thank Ferenbach and colleagues for this important comment on the biases introduced by different single-cell dissociation methods during the single-cell RNA sequencing (scRNA-seq) workflow. An ideal dissociation protocol would generate a cell suspension that accurately reflects the abundance of each cell type in vivo, minimizes artifactual transcriptional responses induced by the dissociation procedure, avoids RNA degradation, and can be applied to cryopreserved tissue. Currently no single protocol accomplishes all of these goals.

Although we agree that nuclear preparations underrepresent leukocytes, enzymatic cell dissociation protocols overrepresent them. In our own human biopsy scRNA-seq dataset relying on enzymatic cell dissociation, 41.4% of the cells were leukocytes.1 But it is unlikely that 40% (in some publications up to 60%) of the cells in a unilateral ureteral obstruction (UUO) kidney are leukocytes because this is typically measured by FACS, which relies on the same enzymatic dissociation protocols that bias against parenchymal cell representation. Indeed, Figure 2 from Eis et al.2 used a histologic examination of F4/80-positive cells, and reported only 14 F4/80-positive cells per high power field, which is about 10% of the cells in that field and four-fold fewer than measured by FACS.

We speculate that nuclear preparations are not simply better at resolving glomerular cell types, but all parenchymal cell types that contain tight junctions and are surrounded by basement membrane. Enzymatic dissociation may lead these epithelia to form clumps that get filtered out of single-cell preparations. This is supported by the observation that our UUO3 dataset of comparable size identified 12 epithelial clusters compared with three epithelial clusters identified in the UUO dataset provided by O’Sullivan et al.4 We also point out the importance of artifactual stress responses induced by dissociation at 37°C when analyzing diseased kidney. In this case deconvoluting artifactual gene expression from appropriate cellular injury responses may be challenging. We agree that applying different dissociation strategies to the same tissue may help resolve the biases intrinsic to scRNA-seq versus single-nucleus RNA sequencing.

Disclosures

None.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

  • See related Letters to the Editor, “Complementary Roles for Single-Nucleus and Single-Cell RNA Sequencing in Kidney Disease Research,” on pages 712–713.

  • Copyright © 2019 by the American Society of Nephrology

References

  1. ↵
    1. Wu H,
    2. Malone AF,
    3. Donnelly EL,
    4. Kirita Y,
    5. Uchimura K,
    6. Ramakrishnan SM, et al
    .: Single-cell transcriptomics of a human kidney allograft biopsy specimen defines a diverse inflammatory response. J Am Soc Nephrol 29: 2069–2080, 2018
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Eis V,
    2. Luckow B,
    3. Vielhauer V,
    4. Siveke JT,
    5. Linde Y,
    6. Segerer S, et al
    .: Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction. J Am Soc Nephrol 15: 337–347, 2004
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Wu H,
    2. Kirita Y,
    3. Donnelly EL,
    4. Humphreys BD
    : Advantages of single-nucleus over single-cell RNA sequencing of adult kidney: Rare cell types and novel cell states revealed in fibrosis. J Am Soc Nephrol 30: 23–32, 2019
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. O'Sullivan ED,
    2. Mylonas JK,
    3. Hughes J,
    4. Ferenbach DA:
    Complementary Roles for Single-Nucleus and Single-Cell RNA Sequencing in Kidney Disease Research. J Am Soc Nephrol 30: 712–713, 2019
    OpenUrlFREE Full Text
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Journal of the American Society of Nephrology: 30 (4)
Journal of the American Society of Nephrology
Vol. 30, Issue 4
April 2019
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Authors’ Reply
Haojia Wu, Yuhei Kirita, Erinn L. Donnelly, Benjamin D. Humphreys
JASN Apr 2019, 30 (4) 714; DOI: 10.1681/ASN.2019020178

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Authors’ Reply
Haojia Wu, Yuhei Kirita, Erinn L. Donnelly, Benjamin D. Humphreys
JASN Apr 2019, 30 (4) 714; DOI: 10.1681/ASN.2019020178
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