Name: Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics (Video) | JoVE Journal
Uploaded: 2024-11-08T12:40:00.000+00:00
Duration: 3 min 39 s
Description: Learn to prepare frozen non-human primate fetal islets for combined single nuclei RNA-seq, ATAC-seq, and bulk metabolomics. This protocol enables multi-layered analysis of gene expression, chromatin accessibility, and metabolite abundance from limited samples.

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5 minutes

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 were necropsied on gestational day 145 (Rhesus macaque gestation is typically 173-175 days). The details of the reagents and the equipment used for this study are listed in the Table of Materials.
1. Islet isolation
<ol>
	<li>Excise fetal pancreata from surrounding tissues9 and place it in ice-cold phosphate-buffered saline.</li>
	<li>Remove the pancreas from PBS and inflate it with collagenase P (0.6 mg/mL) via cannulation of the pancreatic duct using a 33 G rodent brain cannula injector.</li>
	<li>Divide the pancreas into six sections with sterile surgical scissors and digest for 27-30 min at 37 &#176;C in collagenase solution.</li>
	<li>Prepare RT media by combining 10% FBS and 100 U/mL penicillin/streptomycin into RPMI 1640.</li>
	<li>Pour off collagenase and add 10 mL of RT media.</li>
	<li>Gently shake for 1 min to disrupt the remaining connective tissue.</li>
	<li>Remove undigested material by straining through a 500 &#181;m filter.</li>
	<li>Wash the islets twice with 50 mL of RT media.</li>
	<li>Incubate the islets in bovine DNase I (0.8 U/mL) in a 37 &#176;C water bath for 10 min.</li>
	<li>Culture the islets overnight in RT media with bovine DNase I (0.4 U/mL) at 37 &#176;C with 5% CO2.</li>
	<li>Ship islets in specialized packaging at ambient temperature with temperature control gel packs.</li>
</ol>
2. Sample banking for long-term storage
<ol>
	<li>Assess islets for size using a scaled reticle and note the degree of acinar and ductal tissue in preparation.</li>
	<li>Handpick islets with an automated pipette into a 60 mm dish containing 5 mL of 2.8 mM glucose DMEM. 
	NOTE: It is imperative to minimize the amount of acinar tissue and ductal tissue that will be stored in the final islet preparation. Details of ideal islets for use are reported in a previous protocol9.</li>
	<li>Pick islets into a 1.5 mL tube and spin at ~300 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspended the islets in calcium and magnesium-free 1x PBS.</li>
	<li>Spin for 5 min at ~300 x g at 4 &#176;C. Discard the supernatant.</li>
	<li>Repeat steps 2.3-2.5 twice more.</li>
	<li>Flash freeze the islets in a 1.5 mL tube for 10 s in liquid nitrogen.</li>
	<li>Store the pellet at -80 &#176;C until the day of nuclear isolation.</li>
</ol>
3. Cellular fractionation for isolation of nuclei and cytoplasmic fraction
<ol>
	<li>Preparation of buffers for nuclei extraction (Table 1).
	<ol>
		<li>Ensure that the bench, pipettes, and reagents are RNase-free.</li>
		<li>Prepare the wash, concentrated lysis, lysis dilution, and diluted nuclei buffers according to Table 1.</li>
		<li>Prepare 0.1x lysis buffer in a 5 mL tube using the lysis dilution buffer to dilute the concentrated lysis buffer.</li>
	</ol>
	</li>
	<li>Tissue homogenization
	<ol>
		<li>Remove the samples from -80 &#176;C freezer and immediately place them on ice.</li>
		<li>Add 500 &#181;L of 0.1x lysis buffer to the tissue pellets and incubate for 3 min on ice.</li>
		<li>Using a disposable RNase-free pellet pestle, gently homogenize the tissue 15 times on ice.</li>
		<li>Incubate the samples on ice for 10 min.</li>
		<li>Add 500 &#181;L of wash buffer to the homogenate and gently pipette on ice.</li>
		<li>Prime a 30 &#181;m pre-separation filter by adding 300 mL of wash buffer to the filter over a 5 mL tube.</li>
		<li>Filter the 1 mL cellular homogenate using the 30 &#181;M pre-separation filter into the 5 mL tube.</li>
		<li>Spin the filtered homogenate at 500 x g for 5 min in a swinging bucket centrifuge at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Collection and storage of supernatant for metabolomics
	<ol>
		<li>Remove 1 mL of the supernatant from step 3.2.8 with a pipette and add to a 1.5 mL cryovial on ice.</li>
		<li>Immediately freeze the supernatant at -80 &#176;C. This supernatant will be used for metabolomics.</li>
	</ol>
	</li>
	<li>Preparation of nuclei for single nucleus RNA- and ATAC-sequencing
	<ol>
		<li>Gently resuspend the nuclei pellet by adding 1 mL of wash buffer to the pellet dropwise. Slowly pipette 5 times.</li>
		<li>Spin the washed pellet at 1000 x g for 5 min in a swinging bucket centrifuge at 4 &#176;C.</li>
		<li>Repeat steps 3.4.1 and 3.4.2 once more.</li>
		<li>After the final wash, resuspend the nuclei in 1 mL of wash buffer. Keep nuclear preparation on ice.</li>
		<li>Aspirate 10 &#181;L of nuclei suspension with a pipette and mix with 10 &#181;L of 0.4% Trypan blue by gently pipetting.</li>
		<li>Add 10 &#181;L of the nuclei and Trypan blue mixture to an automated cell counter slide with a pipette and place it in the instrument.</li>
		<li>Determine nuclei concentration in nuclei/&#181;L. 
		NOTE: Unlike cellular preparations for single-cell RNA sequencing, automated cell counters will detect nuclei as dead cells. No more than 95% of this preparation should be live on automated cell counters. If more than 5% of the nuclear population is live, the nuclei suspension can be filtered an additional time through the 30 &#181;m pre-separation filter and spun for 5 min at 1000 x g at 4 &#176;C. Nuclei concentration can then be recalculated as outlined in step 3.4.6.</li>
		<li>Based on the desired targeted nuclei recovery, resuspend nuclei to the necessary concentration using 1x nuclei buffer and proceed to single nucleus RNA and ATAC sequencing library preparation10.</li>
	</ol>
	</li>
</ol>
4. Preparation of cytosolic fraction for capillary electrophoresis- mass spectrometry (CE-MS)
<ol>
	<li>Combine 80 &#181;L of cytosolic fraction collected in step 3.3.1 with 20 &#181;L of nanopure water containing internalized standards.</li>
	<li>Proceed with CE-MS and analysis as previously described11,12,13.</li>
</ol>

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

<ol><li>Christensen, A. A., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+beta+cell+in+type+2+diabetes%5BTitle%5D)+AND+%22Curr+Diab+Rep%22%5BJournal%5D)">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/pubmed?term=((Developmental+programming+of+the+pancreatic+islet+by%5BTitle%5D)+AND+%22Trends+Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Risk+factors+and+lifestyle+interventions%5BTitle%5D)+AND+%22Prim+Care%22%5BJournal%5D)">Risk factors and lifestyle interventions.</a> Prim Care. 49 (2), 201-212 (2022).</li>
<li>Pound, L. D., Kievit, P., Grove, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+non-human+primate+as+a+model+for+type+2+diabetes%5BTitle%5D)+AND+%22Curr+Opin+Endocrinol+Diabetes+Obes%22%5BJournal%5D)">The non-human primate as a model for type 2 diabetes.</a> Curr Opin Endocrinol Diabetes Obes. 21 (2), 89-94 (2014).</li>
<li>Kang, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nucleus+RNA+sequencing+of+human+pancreatic+islets+identifies+novel+gene+sets+and+distinguishes+%CE%B2-cell+subpopulations+with+dynamic+transcriptome+profiles%5BTitle%5D)+AND+%22Genome+Med%22%5BJournal%5D)">Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes β-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/pubmed?term=((Using+single-nucleus+RNA-sequencing+to+interrogate+transcriptomic+profiles+of+archived+human+pancreatic+islets%5BTitle%5D)+AND+%22Genome+Med%22%5BJournal%5D)">Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets.</a> Genome Med. 13 (1), 128(2021).</li>
<li>Cabé, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-quality+brain+and+bone+marrow+nuclei+preparation+for+single+nuclei+multiome+assays%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+non-human+primate+pancreatic+islet+oxygen+consumption%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Extraction+of+aqueous+metabolites+from+cultured+adherent+cells+for+metabolomic+analysis+by+capillary+electrophoresis-mass+spectrometry%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Metabolomic+anatomy+of+an+animal+model+revealing+homeostatic+imbalances+in+dyslipidaemia%5BTitle%5D)+AND+%22Mol+Biosyst%22%5BJournal%5D)">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/pubmed?term=((Depiction+of+metabolome+changes+in+histidine-starved+Escherichia+coli+by+CE-TOFMS%5BTitle%5D)+AND+%22Mol+Biosyst%22%5BJournal%5D)">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/pubmed?term=((Single-cell+chromatin+accessibility+identifies+pancreatic+islet+cell+type%E2%80%93+and+state-specific+regulatory+programs+of+diabetes+risk%5BTitle%5D)+AND+%22Nat+Genetics%22%5BJournal%5D)">Single-cell chromatin accessibility identifies pancreatic islet cell type– 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/pubmed?term=((Metaboanalyst+6.0%3A+Towards+a+unified+platform+for+metabolomics+data+processing%2C+analysis+and+interpretation%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Metaboanalyst 6.0: Towards a unified platform for metabolomics data processing, analysis and interpretation.</a> Nucleic Acids Res. , 253(2024).</li>
<li>Willency, J. A., Lin, Y., Pirro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+metabolomics+in+human+and+animal+biofluids+and+tissues+using+liquid+chromatography+coupled+with+tandem+mass+spectrometry%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Targeted metabolomics in human and animal biofluids and tissues using liquid chromatography coupled with tandem mass spectrometry.</a> STAR Protoc. 5 (1), 102884(2024).</li>
<li>Behnke, J. -S., Urner, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emergence+of+mass+spectrometry+detergents+for+membrane+proteomics%5BTitle%5D)+AND+%22Anal+Bioanal+Chem%22%5BJournal%5D)">Emergence of mass spectrometry detergents for membrane proteomics.</a> Anal Bioanal Chem. 415 (18), 3897-3909 (2023).</li>
<li>Ouimet, C. M., D'amico, C. I., Kennedy, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+capillary+electrophoresis+and+the+implications+for+drug+discovery%5BTitle%5D)+AND+%22Expert+Opin+Drug+Discov%22%5BJournal%5D)">Advances in capillary electrophoresis and the implications for drug discovery.</a> Expert Opin Drug Discov. 12 (2), 213-224 (2017).</li>
<li>Alexandrov, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+metabolomics+and+imaging+mass+spectrometry+in+the+age+of+artificial+intelligence%5BTitle%5D)+AND+%22Annu+Rev+Biomed+Data+Sci%22%5BJournal%5D)">Spatial metabolomics and imaging mass spectrometry in the age of artificial intelligence.</a> Annu Rev Biomed Data Sci. 3, 61-87 (2020).</li>
<li>Ewald, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Web-based+multi-omics+integration+using+the+analyst+software+suite%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Web-based multi-omics integration using the analyst software suite.</a> Nat Protoc. 19, 1467-1497 (2024).</li>
</ol>

The authors declare no conflicts of interest.

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 this fractionation with capillary electrophoresis, a technique that requires a smaller sample input relative to liquid chromatography for the separation of metabolites18. 
The most critical aspect of this protocol is the preservation of the nuclear membrane during nuclear isolation to avoid the degradation of genetic material and reduce contamination by ambient nucleotides and macromolecules. Sample exposure times to lysis buffer can be modulated to avoid over-digestion of tissues and nuclei damage. Additionally, quick freezing of the islets during sample banking and the supernatant containing the cytosolic fraction is imperative to avoid the degradation of genetic material and metabolites. All samples must be collected from the same media formulation (2.8 mM glucose DMEM in this study) to avoid the confounding effect of altered metabolism due to nutrient availability in culture media. 
A limitation of this combined metabolomic approach is that some metabolites may not be captured due to the detergent used for nuclei isolation. Therefore, for a more complete metabolite analysis, this approach could be complemented with analyses from frozen tissues and isolating metabolites using non-organic wash buffers and LC-MS. A recommended supplemental approach is targeted spatial metabolomics via MALDI-MS to localize metabolic changes within organs19. Additionally, the present technologies for evaluating transcript variability and chromatin accessibility within the same nucleus are limited due to shallow sequencing depth for ATAC sequencing. In data analyses, investigators will only be able to evaluate open chromatin regions within broad cellular clusters rather than specific subclusters within the dataset. 
As the use of tissues that are not readily available, particularly those from humans and non-human primates, becomes more frequent, it is imperative to maximize the amount of information that can be extracted from a biological replicate. The multi-omic approach described above allows investigators to correlate genetic signatures with peak availability around key promotors of cellular identity while also considering how cellular metabolism may have contributed to such changes. Identifying the metabolites that are more abundant within cellular preparations will give clues to the specific epigenetic modifications that would drive the specification of cellular subtypes to a particular lineage. Indeed, new protocols have been established for integrating the findings of transcriptomic and metabolomic experiments, albeit from different tissue sources20. The significance of this protocol is that chromatin accessibility, transcriptional changes, and metabolite enrichment can be evaluated from a single sample input. Future applications of this technique will allow for better characterization of disease states in tissues acquired from sparse sample populations, such as tissues from patients with rare diseases. In the islet biology field, the characterization of gene regulatory networks and the metabolic signatures that correlate can provide critical insight as to mechanisms of preserving beta-cell identity and function to avoid the onset of diabetes.

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><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&#039;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 &amp;deg;C to dissolve white precipitate</td></tr><tr><td>MACS SmartStrainers (30 &amp;micro;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 &amp;micro;L</td><td>VWR</td><td>76322-528</td><td></td></tr><tr><td>Pipette Tips, 1000 &amp;micro;L</td><td>Thermo Fisher Scientific</td><td>21-236-2A</td><td></td></tr><tr><td>Pipette Tips, 20 &amp;micro;L</td><td>Genesee Scientific</td><td>23-404</td><td></td></tr><tr><td>Pipette Tips, 200 &amp;micro;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)&amp;nbsp;</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

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.

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 contain intact nuclei (Figure 2C).
Details of the analysis of single nucleus RNA- and ATAC-sequencing data are extensively provided elsewhere5,14. Representative clusters of cellular populations from a fetal NHP nuclear preparation are illustrated via UMAP in Figure 3A. Metabolomics data is readily analyzed using open-source software, as previously described15. Generated spectra can be visualized, and enriched metabolites in islets of a fetus from a WSD-fed dam can be compared to those from a CD-fed dam (Figure 3B-F).
<img alt="Simultaneous RNASeq ATACSeq metabolomics; diagram of pancreatic islet isolation, analysis setup." class="xfigimg" src="/files/ftp_upload/66849/66849fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Overview of protocol for isolation of nuclei and cytosolic fraction. Fetal pancreatic islets were banked and stored at -80 °C until the day of the assay. Nuclei were isolated and utilized for single nucleus RNA- and ATAC-Sequencing. The cytosolic fraction following the first wash of nuclei isolation was retained for capillary electrophoresis-mass spectrometry. <a href="https://www.jove.com/files/ftp_upload/66849/66849fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell division process; microscope images; stages A-C; cellular morphology analysis." class="xfigimg" src="/files/ftp_upload/66849/66849fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Demonstration of proper nuclei input into single nucleus multiomics assay. Examples of (A) Nucleus encapsulated by cytoplasm, (B) Bursting nucleus with compromised nuclear membrane, and (C) Intact nucleus with rounded morphology. Scale bars: 10 µm. <a href="https://www.jove.com/files/ftp_upload/66849/66849fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="UMAP clustering map and bar charts comparing metabolite levels (DAG, G3P, 2-OGA, Creatine, GSH)." class="xfigimg" src="/files/ftp_upload/66849/66849fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Representative data from combined single nucleus multiomics and bulk metabolomics. (A) UMAP clustering of cellular subtypes within isolated fetal NHP islets based on gene expression. Quantitative estimation of the glycolytic metabolites, (B) Dihydroxyacetone phosphate and (C) Glycerol-3-phosphate in the cytosolic fraction from isolated fetal NHP islets when the dam was fed a WSD (mWSD) or a CD (mCD). Maternal diet may also differentially regulate metabolism via the (D) citric acid cycle (2-oxoglutarate), (E) amino acid metabolism (creatine), and by altering (F) cellular redox status (glutathione) in fetal islets. <a href="https://www.jove.com/files/ftp_upload/66849/66849fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Buffer formulations for isolation of nuclei. Detailed components of nuclei, lysis, lysis dilution, and wash buffers are shown. If necessary, dilute reagents to the stock concentration before preparing the buffer. <a href="https://www.jove.com/files/ftp_upload/66849/66849_Table%201.xlsx" target="_blank">Please click here to download this Table.</a>

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, ...

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JoVE Journal

Developmental Biology

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Learn to prepare frozen non-human primate fetal islets for combined single nuclei RNA-seq, ATAC-seq, and bulk metabolomics. This protocol enables multi-layered analysis of gene expression, chromatin accessibility, and metabolite abundance from limited samples.

Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics (Video) | JoVE Journal

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

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