Name: preparation of frozen non-human primate fetal islets for combined single nuclei rna-sequencing and atac-sequencing, and bulk metabolomics
Uploaded: 2024-11-08T12:40:00.000+00:00
Duration: 3 min 39 s
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 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

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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. 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. 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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. 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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 ...

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

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 contai...

Author Spotlight: Multi-Layered Approach to Understand Postnatal Functions of Pancreatic Islets in Non-Human Primates

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.

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, ...

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446 Views.  Vanderbilt University. We gebruiken een zeer translationeel, niet-menselijk primatenmodel om te beoordelen hoe maternale voeding en medicatieconsumptie tijdens de zwangerschap de embryonale ontwikkeling en postnatale functie van pancreaseilandjes bij het nageslacht beïnvloeden. Ontwikkelingsgeprogrammeerde veranderingen kunnen de vatbaarheid voor ziekten later in het leven veranderen. Een uitdaging bij het werken met moeilijk te verkrijgen monsters, zoals die van niet-menselijke p...

Video: Auteur in de schijnwerpers: meerlagige benadering om postnatale functies van pancreaseilandjes bij niet-menselijke primaten te begrijpen

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

Cancer Research

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

Genetics

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin ...

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Neuroscience

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Biology

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

Single Cell Nuclei Isolation from IMAT for Studying Gene Expression Profiles 

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

Author Spotlight: Deciphering the Cellular Mysteries of Intermuscular Adipose Tissue in Humans

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose depot.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and ...

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

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