Haojia Wu1, Yuhei Kirita1, Erinn L Donnelly1, Benjamin D Humphreys2,3. 1. Division of Nephrology, Department of Medicine and. 2. Division of Nephrology, Department of Medicine and humphreysbd@wustl.edu. 3. Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
Abstract
BACKGROUND: 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. METHODS: We 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. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. RESULTS: A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation-induced 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. Our 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. CONCLUSIONS: 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.
BACKGROUND: 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. METHODS: We 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. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. RESULTS: A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation-induced 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. Our 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. CONCLUSIONS: 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.
Authors: Jeffrey W Pippin; Matthew A Sparks; Sean T Glenn; Sandra Buitrago; Thomas M Coffman; Jeremy S Duffield; Kenneth W Gross; Stuart J Shankland Journal: Am J Pathol Date: 2013-06-14 Impact factor: 4.307
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Authors: Matthew D Young; Thomas J Mitchell; Felipe A Vieira Braga; Maxine G B Tran; Benjamin J Stewart; John R Ferdinand; Grace Collord; Rachel A Botting; Dorin-Mirel Popescu; Kevin W Loudon; Roser Vento-Tormo; Emily Stephenson; Alex Cagan; Sarah J Farndon; Martin Del Castillo Velasco-Herrera; Charlotte Guzzo; Nathan Richoz; Lira Mamanova; Tevita Aho; James N Armitage; Antony C P Riddick; Imran Mushtaq; Stephen Farrell; Dyanne Rampling; James Nicholson; Andrew Filby; Johanna Burge; Steven Lisgo; Patrick H Maxwell; Susan Lindsay; Anne Y Warren; Grant D Stewart; Neil Sebire; Nicholas Coleman; Muzlifah Haniffa; Sarah A Teichmann; Menna Clatworthy; Sam Behjati Journal: Science Date: 2018-08-10 Impact factor: 63.714