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

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. Overall design: A total of 1000 hand-picked human islets equivalents (IEQs) were transplanted under the kidney capsule of 8-to-12-week-old male Non-obese diabetic (NOD)/SCID-gamma (NSG) mice and animals were followed for 4 weeks. At the end of the follow-up period, mice were sacrificed by cervical dislocation. Human islet grafts were rapidly dissected under the microscope, snap-frozen and stored at -80C. Frozen grafts were transferred to dounce tissue grinder tubes (D8938; Sigma) containing 0.5 ml ice-cold Nuclei EZ lysis buffer (NUC-101; Sigma) and homogenized with pestles A and B on ice each for one minute. Samples were transferred to clean 15 ml tubes and homogenizers were rinsed with 1.5 ml buffer followed by 2 ml buffer and transferred to the same 15 ml tubes to obtain a final volume of 4 ml. The tubes were vortexed briefly at moderate speed and set on ice for 5 minutes for cell lysis. To separate nucleus and cytoplasm, the tubes were centrifuged at 500x g for 5 minutes at 4C. The pellet containing nuclei was resuspended in 0.5 ml cold buffer by vortexing briefly at moderate speed and 3.5 ml cold buffer was added. The nuclear suspension was mixed by vortexing briefly and set on ice for 5 minutes. The tubes were centrifuged at 500x g for 5 minutes and the pellet was resuspended in suspension buffer (0.5 ml PBS containing 0.01% non-acetylated BSA; Sigma and 0.1% RNase inhibitor; 2313A from Clontech). The nuclear suspension was pipetted ten times with a 1 ml tip, filtered through a 30 um pre-separation filter (130-041-407; Miltenyi Biotech); cell number and cell viability were determined by cell counter using 0.4% trypan blue stain. The average number of total nuclei obtained from one-half graft was approximately 8.5x10e5 nuclei (1.7x10e6 cells/ml) with 5-10 um size and 93.3 +/-1.1% dead cell rate (n=32 samples across three independent experiments). The number of isolated nuclei was adjusted to 1000 nuclei/ul with suspension buffer and 10,000 nuclei were immediately used for generation of Gel Beads In-Emulsion (GEMs) and barcoding. Transcriptomic analysis was performed using the 10x Genomics Chromium Single Cell Gene Expression Assay.

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