Abstract
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.
Highlights
Human pancreatic islets are a central focus of research in metabolic studies
Type 1 diabetes (T1D) and type 2 diabetes (T2D) are both characterized by a progressive reduction of functional mass of insulin-producing β-cells [1, 2]
We propose that snRNA-seq is a reliable approach to interrogate the transcriptomic profiles of archived frozen tissues that, to our knowledge, has not been applied previously to pancreatic islets or engrafted tissues
Summary
Human 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 (scRNAseq). A mixture of such diverse cell types can hinder precise identification of cell-specific transcriptomes or exclusive biological signals when bulk tissue analyses are performed To circumvent this limitation, over the past few years, multiple groups have utilized single-cell RNA-seq (scRNA-seq) methodologies on islet cells isolated from mouse [19, 20] or human pancreas [21,22,23,24]. Over the past few years, multiple groups have utilized single-cell RNA-seq (scRNA-seq) methodologies on islet cells isolated from mouse [19, 20] or human pancreas [21,22,23,24] These studies have focused on dissecting the transcriptomic signature of endocrine cell types across different ages [25] and to define differentially expressed genes in type 2 diabetic β-cells [26,27,28,29]. These aspects gain relevance when identifying disease-related transcriptomic signatures in tissues obtained from multiple human donors at different time points that require immediate, usually varied, processing and individual analyses
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