Advantages of single-nucleus over single-cell RNA sequencing

A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses.

Researchers from the Washington University in St. Louis School of Medicine compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney. They validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery.

A total of 11,391 transcriptomes were generated in the comparison phase. The researchers identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociationinduced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. The snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways.

snRNA-seq of day 14 unilateral ureteral obstruction (UUO) kidney identifies rare cell types and and intercellular communication networks

(A, inset) Periodic acid–Schiff stain of UUO kidney showing dilated and cast-filled tubules and expanded and fibrotic interstitium. (A) The t-distributed stochastic neighbor embedding (tSNE) shows 17 separate cell clusters. (B) Projection of cell cycle state onto the tSNE, revealing limited proliferation primarily in the proliferating proximal tubule cluster. (C) Violin plot showing cluster-specific gene expression. (D) Dock10 expression through the proximal tubule but enriched within the dedifferentiating proximal tubule cluster. (E) Three stromal clusters could be identified, including juxtaglomerular apparatus cells expressing Endra and the stem cell marker Hopx. Immunohistochemistry images are from the Human Protein Atlas (https://www.proteinatlas.org/). (F) Cell-specific ligand-receptor analysis reveals intercellular signaling pathways. (G) Known and new intercellular signaling within the tubulintersitial compartment as revealed by this snRNA-seq analysis. Act., activating; CD-PC, collecting duct-principal cell; CNT, connecting tubule; DCT, distal convoluted tubule; Dediff., dedifferentiated; DL + tAL, descending limb + thin ascending limb; EC, endothelial cell; Fib., fibroblast; IC, intercalated cell; JGA, juxtaglomerular apparatus; MΦ, macrophage; PC, principal cell; Pod, podocyte; Prolif, proliferating; PT, proximal tubule; TAL, thick ascending limb; UMI, unique molecular identifier.

snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney.

Wu H, Kirita Y, Donnelly EL, Humphreys BD. (2021) 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(1):23-32. [article]

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