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.