Ferenbach, David A.

Hughes, Jeremy

Mylonas, Katie J.

O’sullivan, Eoin D.

Edinburgh Research Explorer

Letter to the Editors:Complementary roles for single nucleus and single cell RNA sequencing in kidney disease research.Eoin D O’Sullivan MD 1,2, Katie J Mylonas PhD2 and David A Ferenbach MD PhD1,21Department of Renal Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK  2Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK.  3Renal Division and Division of Engineering in Medicine, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.Dr David Ferenbach, Centre for Inflammation Research,Queen’s Medical Research Institute,47 Little France Crescent, Edinburgh, EH16 4TJ, UK. Email : david.ferenbach@ed.ac.ukRunning title: Single nucleus and single cell sequencing  Integrated DiscussionWe congratulate Wu et al. on their successful application of single nucleus RNA sequencing to characterise renal cell types underrepresented by single cell RNA sequencing.1 A particular strength of their workflow is the detection of cells such as podocytes, mesangial cells, and glomerular endothelial cells, which can be underrepresented by conventional single-cell sequencing. These cell types have previously been described as distinct clusters using single-cell sequencing at markedly higher total cell numbers, such as the recent landmark renal single-cell sequencing paper by Park et al 2As a group characterizing renal injuries using a similar single-cell, high-throughput, droplet-based platform, we are struck by the differences in cell classification between those described by Wu et al.using single-nucleus sequencing and those seen in our own single-cell sequencing of similar kidneys after unilateral ureteric obstruction (UUO). In contrast to the authors, we detect multiple leukocytes in our samples and identify clusters of distinct subsets of mononuclear phagocytes (35%), neutrophils (1.8%), B lymphocytes (3.3%), and T lymphocytes (10%) from a library of 6213 transcriptomes (Figure 1). This correlates well with existing histological studies and FACS analysis of digested UUO kidneys showing nearly 40% of renal cells are CD45+ve leukocytes3, 4 5 and is in contrast to a single small macrophage cluster found on the single nucleus analysis.  Several groups have reported the importance of both T and B lymphocytes in the pathogenesis of UUO, yet single-nuclear RNA sequencing seems not to identify such cells, with representative lymphocyte marker genes suggested by Park et al. instead expressed in sparse cells scattered across multiple clusters in the single-nucleus dataset.It is probable that differing technologies may introduce a cell-specific bias into the library preparation. Single-nucleus sequencing appears to bring improved identification of glomerular cell types. Single-cell sequencing may be more biased toward leukocytes, to the detriment of those glomerular cells. The advantages of single-nucleus sequencing are important and include the reduction of stress-response gene signatures and the ability to sequence historical frozen samples. However, we wonder if current protocols lack the ability to characterize the full spectrum of leukocytes present within the kidney. We propose that complimentary roles exist for both single-cell and single-nucleus sequencing, and researchers may wish to consider the cell-type bias in any workflow, and their own cell of interest, when designing sequencing experiments. AcknowledgementsAll authors declare no competing financial interests.Figure legend. T-SNE plot demonstrating distinct shared nearest neighbour cluster formation, annotated by cell type B) Leukocyte gene expression projected across these T-SNE clusters C) Leukocyte genes expression plotted across clusters, natural logarithmic scale. D) Cell frequency as percentage of total library sizeReferences1. Wu, H, Kirita, Y, Donnelly, EL, 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, 2018.2. Park, J, Shrestha, R, Qiu, C, Kondo, A, Huang, S, Werth, M, Li, M, Barasch, J, Susztak, K: Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease. Science, 2018.3. Eis, V, Luckow, B, Vielhauer, V, Siveke, JT, Linde, Y, Segerer, S, Perez De Lema, G, Cohen, CD, Kretzler, M, Mack, M, Horuk, R, Murphy, PM, Gao, JL, Hudkins, KL, Alpers, CE, Grone, HJ, Schlondorff, D, Anders, HJ: Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction. J Am Soc Nephrol, 15: 337-347, 2004.4. Liu, L, Kou, P, Zeng, Q, Pei, G, Li, Y, Liang, H, Xu, G, Chen, S: CD4+ T Lymphocytes, especially Th2 cells, contribute to the progress of renal fibrosis. Am J Nephrol, 36: 386-396, 2012.5. Yang, J, Zhu, F, Wang, X, Yao, W, Wang, M, Pei, G, Hu, Z, Guo, Y, Zhao, Z, Wang, P, Mou, J, Sun, J, Zeng, R, Xu, G, Liao, W, Yao, Y: Continuous AMD3100 Treatment Worsens Renal Fibrosis through Regulation of Bone Marrow Derived Pro-Angiogenic Cells Homing and T-Cell-Related Inflammation. PLoS One, 11: e0149926, 2016.Figure 1

Complementary Roles for Single-Nucleus and Single-Cell RNA Sequencing in Kidney Disease Research

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