<meta charset="utf8"></head><body>Von der RNA zu den Metaboliten: Multi-Omics-Profiling von nicht-menschlichen Primateninseln

<ol>
	<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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+non-human+primate+as+a+model+for+type+2+diabetes.">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/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. A., Lin, Y., Pirro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+metabolomics+in+human+and+animal+biofluids+and+tissues+using+liquid+chromatography+coupled+with+tandem+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+mass+spectrometry+detergents+for+membrane+proteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+capillary+electrophoresis+and+the+implications+for+drug+discovery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+metabolomics+and+imaging+mass+spectrometry+in+the+age+of+artificial+intelligence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+multi-omics+integration+using+the+analyst+software+suite.">Web-based multi-omics integration using the analyst software suite.</a> Nat Protoc. 19, 1467-1497 (2024).</li></ol>

<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

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.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Mehrschichtiger Ansatz zum Verständnis der postnatalen Funktionen von Pankreasinseln bei nicht-menschlichen Primaten

Aufbereitung von gefrorenen nicht-menschlichen Primateninseln für die kombinierte Einzelkern-RNA-Sequenzierung und ATAC-Sequenzierung sowie Bulk-Metabolomik

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

Research

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JoVE Journal

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

Developmental Biology

242 Aufrufe.  Vanderbilt University. Wir verwenden ein hochtranslationales, nicht-humanes Primatenmodell, um zu untersuchen, wie die Ernährung der Mutter und der Medikamentenkonsum während der Schwangerschaft die Embryonalentwicklung und die postnatale Funktion der Pankreasinseln bei den Nachkommen beeinflussen. Entwicklungsbedingt programmierte Veränderungen könnten die Anfälligkeit für Krankheiten im späteren Leben verändern. Eine Herausforderung bei der Arbeit mit schwer zu beschaffen...

Serie: Autor im Rampenlicht: Mehrschichtiger Ansatz zum Verständnis der postnatalen Funktionen von Pankreasinseln bei nicht-menschlichen Primaten

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.

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.