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Gradient-Free Isolation of Murine Pancreatic Islets for Single-Cell RNA Sequencing

ABSTRACT: Pancreatic islet isolation for single-cell RNA sequencing (scRNA-seq) requires extensive enzymatic and mechanical processing that may affect cell recovery and transcriptional state. We evaluated a practical gradient-free workflow for murine pancreatic islets from diabetic BKS.Cg-Dock7^m +/+ Lepr^db/J mice. The protocol combined intraductal collagenase perfusion, controlled digestion, stereomicroscope-guided manual islet selection without density-gradient purification, and gentle Accutase/EDTA dissociation. Compared with Ficoll-based purification, the gradient-free workflow omitted density-gradient preparation, gradient centrifugation, additional wash centrifugations, and associated transfer steps while yielding viable cells suitable for scRNA-seq. The resulting library met the applied QC criteria and retained the major endocrine populations. Stress-associated transcriptional differences between the two pooled libraries were descriptive only. Overall, the gradient-free workflow represents a feasible alternative to Ficoll-based purification for preparing murine pancreatic islets for scRNA-seq.

ORGANISM(S): Mus musculus

PROVIDER: GSE347182 | GEO | 2026/09/30

REPOSITORIES: GEO

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Project description:Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis. Examination of the miRNA profiles in 3 preparations of isolated pancreatic islets and 3 preparations of FACS-enriched pancreatic beta-cells

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Gradient-Free Isolation of Murine Pancreatic Islets for Single-Cell RNA Sequencing

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