Name: Author Spotlight: Multi-Layered Approach to Understand Postnatal Functions of Pancreatic Islets in Non-Human Primates
Uploaded: 2024-11-08T12:40:00
Description: One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, ...

The authors would like to thank the entire team at the Oregon Health Sciences University for taking care of the non-human primate colony and for tissue acquisition. Additionally, Vanderbilt Technologies for Advanced Genomics provided technical expertise and experimental design advice. DTC was supported by an F31 pre-doctoral fellowship from the NIH/NIDK (1F31DK135164). PK was supported by the NIH/NIDDK (1R01DK128187). MG was supported by a VA Merit award (I01 BX005399), the NIH/NIDDK (1R01DK135032, 1R01DK128187), and the JDRF (2-SRA-2024-1455-S-B). Work at the Oregon National Primate Research Center (ONPRC) was supported by P51OD011092 for operational support of the Center.

Islet retrieval was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Oregon National Primate Research Center (ONPRC) and Oregon Health and Science University and was approved by the ONPRC IACUC. The ONPRC abides by the Animal Welfare Act and Regulations enforced by the United States Department of Agriculture (USDA) and the Public Health Service Policy on Humane Care and Use of Laboratory. The protocol herein was applied to islets from fetal Rhesus macaques that...

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

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	<li>Christensen, A. A., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+cell+in+type+2+diabetes.">The beta cell in type 2 diabetes.</a> Curr Diab Rep. 19 (9), 81 (2019).</li><li>Elsakr, J. M., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+programming+of+the+pancreatic+islet+by.">Developmental programming of the pancreatic islet by.</a> Trends Dev Biol. 10, 79-95 (2017).</li><li>Salzberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+and+lifestyle+interventions.">Risk factors and lifestyle interventions.</a> Prim Care. 49 (2), 201-212 (2022).</li><li>Pound, L. D., Kievit, P., Grove, K. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleus+RNA+sequencing+of+human+pancreatic+islets+identifies+novel+gene+sets+and+distinguishes+&#946;-cell+subpopulations+with+dynamic+transcriptome+profiles.">Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes &#946;-cell subpopulations with dynamic transcriptome profiles.</a> Genome Med. 15 (1), 30 (2023).</li><li>Basile, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-nucleus+RNA-sequencing+to+interrogate+transcriptomic+profiles+of+archived+human+pancreatic+islets.">Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets.</a> Genome Med. 13 (1), 128 (2021).</li><li>Cab&#233;, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+brain+and+bone+marrow+nuclei+preparation+for+single+nuclei+multiome+assays.">High-quality brain and bone marrow nuclei preparation for single nuclei multiome assays.</a> J Vis Exp. (202), e65715 (2023).</li><li>. Nuclei from embryonic mouse brain for single cell multiome ATAC + gex sequencing Available from: <a target="_blank" href="https://www.10xgenomics.com">https://www.10xgenomics.com</a> (2022)</li><li>Elsakr, J. M., Deeter, C., Ricciardi, V., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+non-human+primate+pancreatic+islet+oxygen+consumption.">Analysis of non-human primate pancreatic islet oxygen consumption.</a> J Vis Exp. (154), e60696 (2019).</li><li>. Chromium next gem single cell multiome ATAC + gene expression reagent kits user guide Available from: <a target="_blank" href="https://www.10xgenomics.com">https://www.10xgenomics.com</a> (2022)</li><li>Maruyama, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+aqueous+metabolites+from+cultured+adherent+cells+for+metabolomic+analysis+by+capillary+electrophoresis-mass+spectrometry.">Extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis by capillary electrophoresis-mass spectrometry.</a> J Vis Exp. (148), e59551 (2019).</li><li>Ooga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+anatomy+of+an+animal+model+revealing+homeostatic+imbalances+in+dyslipidaemia.">Metabolomic anatomy of an animal model revealing homeostatic imbalances in dyslipidaemia.</a> Mol Biosyst. 7 (4), 1217-1223 (2011).</li><li>Ohashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depiction+of+metabolome+changes+in+histidine-starved+Escherichia+coli+by+CE-TOFMS.">Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS.</a> Mol Biosyst. 4 (2), 135-147 (2008).</li><li>Chiou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+chromatin+accessibility+identifies+pancreatic+islet+cell+type&#8211;+and+state-specific+regulatory+programs+of+diabetes+risk.">Single-cell chromatin accessibility identifies pancreatic islet cell type&#8211; and state-specific regulatory programs of diabetes risk.</a> Nat Genetics. 53 (4), 455-466 (2021).</li><li>Pang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaboanalyst+6.0:+Towards+a+unified+platform+for+metabolomics+data+processing,+analysis+and+interpretation.">Metaboanalyst 6.0: Towards a unified platform for metabolomics data processing, analysis and interpretation.</a> Nucleic Acids Res. , 253 (2024).</li><li>Willency, J. 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Historically, the methods of nuclear isolation for single nucleus RNA- and ATAC-sequencing have utilized buffers containing detergents that are incompatible with liquid chromatography-mass spectrometry (LC-MS), a common technique used for metabolite quantification16,17. The current protocol allows for the combination of transcriptomic and metabolomic techniques from the same sample preparation by using a gentle detergent for cellular fractionation, and following ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>33 G rat brain cannula injector</td>
			<td>Protech International</td>
			<td>8IC315IS5SPC</td>
			<td>For duct cannulation and pancreas inflation</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>15-140-122</td>
			<td>For RT media</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Millipore/Sigma</td>
			<td>F4135-500ML</td>
			<td>For RT media</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-875-135</td>
			<td>For RT media</td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma Aldrich</td>
			<td>11213865001</td>
			<td>For digestion of pancreas</td>
		</tr>
		<tr>
			<td>Bovine Dnase I</td>
			<td>Sigma Aldrich</td>
			<td>11284932001</td>
			<td>For RT media and isolation</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D16-1</td>
			<td>Final concentration will be 2.8 mM</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher</td>
			<td>C4901-500G</td>
			<td>Final concentration will be 0.5 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Final concentration will be 10 mM</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) Fraction 5</td>
			<td>Research Products International</td>
			<td>A30075100</td>
			<td>Final concentration will be 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11966025</td>
			<td>For handpicking islets</td>
		</tr>
		<tr>
			<td>20x Nuclei Buffer</td>
			<td>10X Genomics</td>
			<td>2000153/2000207</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin (5%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BN2006</td>
			<td>Digitonin may need to be warmed to 65 &#176;C to dissolve white precipitate</td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (30 &#181;M)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution (10%)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma Aldrich</td>
			<td>i8896</td>
			<td>Prepare a 10% stock using MilliQ water</td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride Solution, pH 7.5</td>
			<td>Sigma Aldrich</td>
			<td>T2319-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride Solution, 5 M</td>
			<td>Sigma Aldrich</td>
			<td>S6546-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution, 1 M</td>
			<td>Sigma Aldrich</td>
			<td>M1028-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma Protector Rnase inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Research Products International</td>
			<td>D11000-5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase-Free Disposable Pellet Pestles</td>
			<td>Fisher Scientific</td>
			<td>12-141-368</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Bioreagents</td>
			<td>BP337-500</td>
			<td>Prepare a 20% stock using MilliQ water</td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Promega</td>
			<td>PR-P1193</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes (5 mL)</td>
			<td>Eppendorf</td>
			<td>30108310</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials (1.5 mL)</td>
			<td>VWR</td>
			<td>20170-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click Tubes (0.6 mL)</td>
			<td>Denville</td>
			<td>C-2177</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue (0.4%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>T-10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Chamber Cell Counting Slides</td>
			<td>Bio-Rad</td>
			<td>1450011</td>
			<td></td>
		</tr>
		<tr>
			<td>MiiliQ Ultrapure Water System</td>
			<td>Millipore/Sigma</td>
			<td>7003/05/10/15</td>
			<td></td>
		</tr>
		<tr>
			<td>5 kDa cut-off filter</td>
			<td>HMT Metabolome Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Standards</td>
			<td>HMT Metabolome Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P10, P20, P200, and P1000 Pipettes</td>
			<td>Eppendorf</td>
			<td>2231000602</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 10 &#181;L</td>
			<td>VWR</td>
			<td>76322-528</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 1000 &#181;L</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-236-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 20 &#181;L</td>
			<td>Genesee Scientific</td>
			<td>23-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 200 &#181;L</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase Contrast Microscope</td>
			<td>Olympus</td>
			<td>BX41-PH-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1x)&#160;</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-30R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>B06315</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-ZAP</td>
			<td>Sigma Aldrich</td>
			<td>R2020</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of frozen non-human primate fetal islets for combined single nuclei rna-sequencing and atac-sequencing, and bulk metabolomics

One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.

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.

The quality of the sequencing outputs is highly dependent on the quality of the nuclear input. Nuclear blebbing and damage to the outer nuclear membrane will result in ambient RNA and DNA contamination in the analysis, which will introduce noise in the data. A pure nuclear extract will have minimal evidence of nuclei encapsulated with cytoplasmic components (Figure 2A) or overdigestion, evidenced by bursting nuclei (Figure 2B). An ideal nuclear input will contai...

This protocol describes procedures to isolate high-quality nuclei from frozen non-human primate pancreatic islets for use in single-nucleus simultaneous RNA sequencing and ATAC sequencing while preserving the bulk cytosolic fraction for metabolomic analyses from the same samples.

One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, ...

preparation-frozen-non-human-primate-fetal-islets-for-combined-single

Without Section

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.