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 primate 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 reagent and 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.

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 RNA's 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 500 G for five minutes in a swinging bucket centrifuge at 4 degrees Celsius.

Using a pipette, transfer one milliliter of the supernatant to a cryovial on ice and immediately freeze it at 80 degrees Celsius for future metabolomics experiments. Add one milliliter of wash buffer dropwise to the nuclei pellet to resuspend it. Spin the washed pellet at 1000 G for five minutes in a swinging bucket centrifuge at 4 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-oxoglutaric, creatine, and glutathione was obtained using the cytosolic fractions.