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Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets

ABSTRACT: Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets

PROVIDER: PRJNA631512 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11746586_1.fastq.gz	Fastqsanger.gz
	SRR11746586_2.fastq.gz	Fastqsanger.gz
	SRR11746587_1.fastq.gz	Fastqsanger.gz
	SRR11746587_2.fastq.gz	Fastqsanger.gz
	SRR11746588_1.fastq.gz	Fastqsanger.gz

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Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets

Project description:Human pancreatic islets, including insulin secreting beta-cells are a major focus of transplantation strategies aimed at identifying new therapeutic approaches to counteract hyperglycemia in patients with diabetes. Identifying the transcriptomic signature of human islet cells provides insights into regulatory pathways that can be harnessed for planning therapeutic strategies. In this context, single-cell RNA-sequencing (scRNA-seq) has been used mostly in vitro. However, in experimental human islet transplantation models the small amount of tissue is principally used for immunostaining and poses a challenge in performing ‘omics’ studies that provide unbiased information. To circumvent this limitation, we report the use of single nucleus RNA-sequencing (snRNA-seq) on frozen/archived human islet grafts, to define the transcriptomic signature of islet cells preserved after in vivo studies. Interrogating nuclear RNA, we were able to successfully identify all islet endocrine cells, obtain adequate coverage of genes and define molecular pathways that are important for studying human islet cell biology (e.g. cell cycle, apoptosis, insulin secretion). Intersecting our nuclear transcriptomic output with publicly available single-cell RNA-seq datasets, revealed ~90% overlap of the detected genes. In conclusion, we propose that snRNA-seq represents a reliable strategy to probe transcriptomic profiles of fresh or archived transplanted human islets.

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Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets.

Project description:BackgroundHuman pancreatic islets are a central focus of research in metabolic studies. Transcriptomics is frequently used to interrogate alterations in cultured human islet cells using single-cell RNA-sequencing (scRNA-seq). We introduce single-nucleus RNA-sequencing (snRNA-seq) as an alternative approach for investigating transplanted human islets.MethodsThe Nuclei EZ protocol was used to obtain nuclear preparations from fresh and frozen human islet cells. Such preparations were first used to generate snRNA-seq datasets and compared to scRNA-seq output obtained from cells from the same donor. Finally, we employed snRNA-seq to obtain the transcriptomic profile of archived human islets engrafted in immunodeficient animals.ResultsWe observed virtually complete concordance in identifying cell types and gene proportions as well as a strong association of global and islet cell type gene signatures between scRNA-seq and snRNA-seq applied to fresh and frozen cultured or transplanted human islet samples.ConclusionsWe propose snRNA-seq as a reliable strategy to probe transcriptomic profiles of freshly harvested or frozen sources of transplanted human islet cells especially when scRNA-seq is not ideal.

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