cellular fractionation of rhesus macaque pancreatic islets for single-cell rna sequencing and metabolomics

From RNA to Metabolites: Multi-Omic Profiling of Non-Human Primate Fetal Islets

The pancreatic islets of Langerhans are critical for the management of glucose tolerance throughout the lifespan. Failure to properly tune blood glucose levels via the glucose-lowering hormone, insulin, or the glucose-raising hormone, glucagon results in Diabetes Mellitus1. Modifiable risk factors to minimize diabetes risk include exercise and diet. In addition, it is becoming clear that maternal diet during pregnancy also has an effect on offspring susceptibility to diabetes in adulthood2,3. Thus, methods are needed to analyze the effects of maternal diet and other exposures, such as medications used during pregnancy, on the insulin-producing beta cells in model organisms during development. For example, islets from fetal non-human primate (NHP) offspring can be evaluated for the effects of developmental exposures. Similar to humans, NHPs typically have singleton births and vary in their physiological response to Western-style diet feeding4. While the gestation and fetal development of NHPs shares many similarities with humans, making them a highly relevant species for metabolic programming studies, challenges to performing comparative bioinformatic assays on NHP offspring include a restricted mating season and intermittent sample collection. Therefore, approaches that allow for long-term storage prior to sample processing and analysis are ideal. This protocol describes the preparation of samples for single nucleus RNA-sequencing, ATAC-sequencing, and bulk cytoplasmic metabolomics.
Studies by others in the islet biology field have demonstrated overlap in the genetic signatures observed in samples prepared via single-cell RNA Sequencing and single-nucleus RNA sequencing5,6. Both methods allow for the identification of transcripts that contribute to distinguishing features amongst various cell types within the islet population. Through the use of gentle detergents on flash-frozen tissue, intact nuclei can simultaneously be used for ATAC sequencing, which allows for the determination of accessible chromatin regions. Combining single-nucleus RNA-sequencing with ATAC-sequencing allows for better characterization of gene regulatory networks7.
The preparation of samples for metabolomics has traditionally been limited by the need for a high sample volume input for liquid chromatography. In addition, careful consideration must be given to the buffers used during sample preparation to avoid false NMR peaks in mass spectrometric data. Given the low number of samples that are challenging and costly to acquire in primate studies, allocation of islets from different offspring toward transcriptomic versus metabolomic experiments can be difficult. Thus, this protocol makes use of the normally discarded cytosolic fraction generated during cellular fractionation for single-nucleus experiments. This method allows for the simultaneous characterization of transcripts and open chromatin regions that define cellular subsets in fetal islets while quantitatively measuring the metabolites produced within islets (Figure 1). This protocol is adapted from a demonstrated protocol published by 10x genomics for the isolation of nuclei from flash-frozen embryonic mouse brain tissue (version CG000366 Rev D8)7.

Author Spotlight: Multi-Layered Approach to Understand Postnatal Functions of Pancreatic Islets in Non-Human Primates

Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics

We utilize a highly translational, non-human primate model to assess how maternal diet and medication consumption during pregnancy influence the embryonic development and postnatal function of pancreatic islets in the offspring. Developmentally programmed changes could alter disease susceptibility later in life. A challenge when working with hard-to-acquire samples such as those from non-human primates is evaluating multiple questions from the same individual efficiently.Unfortunately, simultaneously extracting information from the metabolome and genome can be difficult due to the incompatibility of assay reagents in time-sensitive degradation of molecules to be quantified. Our protocol allows for extrapolation of metabolite abundance, chromatin accessibility, and differential gene expression from the same starting material. This multi-layered approach provides insight into how metabolism may alter the genome in pancreatic islets.

Cellular Fractionation of Rhesus Macaque Pancreatic Islets for Single-Cell RNA Sequencing and Metabolomics

To begin, thaw the isolated rhesus macaque pancreatic islet tissue on ice. Add 500 microliters of 0.1X lysis buffer to the tissue pellet. Using a disposable RNase-free pellet pestle, gently homogenize the tissue 15 times on ice.Incubate the sample on ice for 10 minutes. Then, add 500 microliters of wash buffer to the homogenate, and gently mix with pipette on ice. Prime a 30-micrometer pre-separation filter with 300 milliliters of wash buffer, and filter one milliliter of cellular homogenate into a five-milliliter tube.Spin the filtered homogenate at 500g for five minutes in a swinging bucket centrifuge at four degrees Celsius. Using a pipette, transfer one milliliter of the supernatant to a cryovial on ice, and immediately freeze it at minus 80 degrees Celsius for future metabolomics experiments. Add one milliliter of wash buffer drop-wise to the nuclei pellet to resuspend it.Spin the washed pellet at 1, 000g for five minutes in a swinging bucket centrifuge at four degrees Celsius. After repeating the wash, resuspend the nuclei in one milliliter of wash buffer. With a pipette, mix 10 microliters each of nuclei suspension and 0.4%Trypan Blue in a separate tube, and add the mixture to an automated cell counter slide.Finally, using a cell counter, determine the nuclei concentration. An ideal intact nucleus was obtained for the sequencing experiments. Based on gene expression studies, the UMAP clustering of cellular subtypes within isolated fetal, non-human, primate islets was obtained.Islet metabolite abundance, such as dihydroxyacetone phosphate, glycerol 3-phosphate, 2-oxoglutarate, creatine, and glutathione was obtained using the cytosolic fractions.

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