We would like to thank David Reynolds from the Albert Einstein Genomics core and Jinghang Zhang from the Flow Cytometry Core for technical support. We acknowledge support from the National Institutes of Health (NIH) (DK110426) and Pilot and Feasibility Grants from the Einstein-Mount Sinai Diabetes Research Center (DK020541), and New York Obesity Research Center (DK026687) (all to K.S.). We also would like to thank Albert Einstein Cancer Center (CA013330) for core support.

Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine.
1. Preparation of tissue digestion and lysis buffers
<ol>
	<li>Prepare tissue digestion buffer.
	<ol>
		<li>Prepare ~1 mL of digestion buffer for every gram of adipose tissue.</li>
		<li>Weigh out 1.5 U/mL collagenase D and 2.4 U/mL dispase II and add phosphate buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Prepare nuclei preparation buffer (NPB).
	<ol>
		<li>Prepare 10 mM HEPES, 1.5 mM magnesium chloride, 10 mM potassium chloride, 250 mM sucrose, and 0.1% NP-40 in nuclease-free water. Mix well. Sucrose may take more time to dissolve than the other components.</li>
	</ol>
	</li>
	<li>Prepare 0.5% bovine serum albumin (BSA) solution.
	<ol>
		<li>Prepare 0.5% BSA in PBS containing EDTA. It is recommended to use sterile-filtered cell culture grade PBS (see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Enzymatic digestion of adipose tissue
<ol>
	<li>Dissect mice to extract adipose tissue according to Aune et al.13. Collect isolated tissue in a dish containing PBS. Transfer the tissue to a paper towel to pat dry. Then place the tissue in a clean dish.</li>
	<li>Add calcium chloride to the digestion buffer to a final concentration of 10 mM. Add a small volume of digestion buffer to the dish and thoroughly mince the adipose tissue. The volume of digestion buffer required will be based on the amount of tissue isolated. Usually ~1 mL or less is used.</li>
	<li>Add about half of the amount of prepared digestion buffer (e.g., ~10 mL for five mice) to the minced tissue. Using a serological pipette, transfer the sample to a 50 mL conical centrifuge tube. Add any remaining amount of buffer to wash the dish and transfer to the 50 mL tube containing the sample. Pipette up and down several times. Then incubate with shaking at 200-210 rpm at 37 &#176;C for 12-15 min.</li>
</ol>
3. Adipocyte isolation
<ol>
	<li>After incubation, add 0.5% BSA at a 1:1 ratio to the sample. Mix well by pipetting up and down several times. Centrifuge the sample at 300 x g for 5 min at room temperature (RT) to spin down the stromal vascular fraction (SVF). The adipocyte fraction will remain in the top layer of the sample.</li>
	<li>Place a 40 &#181;m cell filter on top of a 50 mL conical centrifuge tube and filter the sample, collecting the top layer and leaving behind the SVF at the bottom of the tube. Transfer the top layer (adipocyte fraction) to a new tube by flipping the filter upside down over the tube and using 0.5% BSA to reverse wash the filter and collect the sample in the tube.</li>
	<li>Bring the total volume of 0.5% BSA to 10-15 mL based on sample size and pipette up and down with a serological pipette or briefly shake up and down to mix the sample. Centrifuge the sample again at 300 x g for 5 min at RT.</li>
</ol>
4. Nuclei isolation
<ol>
	<li>Use a wide-bore pipette tip and carefully transfer the top layer of the sample to a 100 &#181;m cell filter placed on top of a new prechilled tube on ice, leaving behind any residual SVF.</li>
	<li>Rinse/wash the filter with a sufficient amount of NPB and discard the filter. From this step forward have the sample on ice as often as possible and work quickly to avoid leaky and/or unhealthy nuclei. It is helpful to prechill microcentrifuge tubes to be used in future steps on ice.</li>
	<li>Place the sample on ice for no more than 2 min with intermittent gentle inverting of the tube to mix. The incubation time on ice should be optimized for sample size and type of adipocytes. Centrifuge at 1,000 x g at 4 &#176;C for 10 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of NPB. Transfer the sample to a clean microcentrifuge tube. While transferring, avoid touching the walls of the tube, which contain debris and lipid. Add 0.6 U/&#181;L RNase inhibitor to the sample and mix. Centrifuge again at 1,000 x g at 4 &#176;C for 10 min. 
	NOTE: Centrifugation speed and time should be decreased at this step for smaller samples.</li>
	<li>Remove the supernatant and resuspend in 1 mL of Nuclei Wash Buffer (2% BSA/PBS + 0.6 U/&#181;L RNase inhibitor).</li>
	<li>Double filter into a clean tube with stackable 30 &#181;m filters. Wash filter with a small volume (~300 &#181;L) of Nuclei Wash Buffer. Alternatively, for smaller samples, use a 30 &#181;m filter that fits into a microcentrifuge tube and filter directly into it.</li>
	<li>Confirm successful isolation of healthy nuclei.
	<ol>
		<li>Stain a small aliquot of the sample with trypan and use an automated cell counter to assess average dead size and percent dead cells. Average dead size for a sample containing mostly nuclei should range between 7-10 &#181;m. Nuclei size is usually around 8 &#181;m.</li>
		<li>Stain a small aliquot of the sample with DAPI and examine under a microscope with a 20x objective or higher. The nuclear membrane should be intact and the nuclei should be round.</li>
	</ol>
	</li>
</ol>
5. FACS cleanup and nuclei concentration step
<ol>
	<li>Proceed immediately to the FACS step to clean up nuclei and remove any excess debris. The collection buffer should contain Nuclei Wash Buffer. During FACS the buffer is diluted. Preparing a higher concentration of BSA and RNase inhibitor to account for dilution during sorting is helpful. The FACS collection block should be on cooling mode.</li>
	<li>After sorting the nuclei, centrifuge at 500 x g for 5 min at 4 &#176;C to concentrate the sample. Reconstitute in an appropriate volume of Nuclei Wash Buffer to achieve a concentration of 500-1,500 nuclei/&#181;L. Do not attempt to remove all the supernatant. Doing so will result in loss of nuclei.</li>
	<li>Use a hemocytometer and a DAPI stained aliquot for a final count and to visualize the nuclei. Then proceed immediately with snRNA-seq according to the manufacturer&#39;s protocol.</li>
</ol>

<ol>
	<li>Cypess, A. 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=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> The New England Journal of Medicine. 360 (15), 1509-1517 (2009).</li><li>van Marken Lichtenbelt, W. 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=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> The New England Journal of Medicine. 360 (15), 1500-1508 (2009).</li><li>Virtanen, K. 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=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> The New England Journal of Medicine. 360 (15), 1518-1525 (2009).</li><li>Nedergaard, J., Bengtsson, T., Cannon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+evidence+for+active+brown+adipose+tissue+in+adult+humans.">Unexpected evidence for active brown adipose tissue in adult humans.</a> American Journal of Physiology-Endocrinology and Metabolism. 293 (2), E444-E452 (2007).</li><li>Saito, 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+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58 (7), 1526-1531 (2009).</li><li>Yoneshiro, 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=Recruited+brown+adipose+tissue+as+an+antiobesity+agent+in+humans.">Recruited brown adipose tissue as an antiobesity agent in humans.</a> The Journal of Clinical Investigation. 123 (8), 3404-3408 (2013).</li><li>Cypess, A. 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=Activation+of+human+brown+adipose+tissue+by+a+&#946;3-adrenergic+receptor+agonist.">Activation of human brown adipose tissue by a &#946;3-adrenergic receptor agonist.</a> Cell Metabolism. 21 (1), 33-38 (2015).</li><li>Song, 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=Low-+and+high-thermogenic+brown+adipocyte+subpopulations+coexist+in+murine+adipose+tissue.">Low- and high-thermogenic brown adipocyte subpopulations coexist in murine adipose tissue.</a> The Journal of Clinical Investigation. 130 (1), 247-257 (2020).</li><li>Cinti, S., 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=CL316,243+and+cold+stress+induce+heterogeneous+expression+of+UCP1+mRNA+and+protein+in+rodent+brown+adipocytes.">CL316,243 and cold stress induce heterogeneous expression of UCP1 mRNA and protein in rodent brown adipocytes.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 50 (1), 21-31 (2002).</li><li>Chen, 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=Thermal+stress+induces+glycolytic+beige+fat+formation+via+a+myogenic+state.">Thermal stress induces glycolytic beige fat formation via a myogenic state.</a> Nature. 565 (7738), 180-185 (2019).</li><li>Bakken, T. E., 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+and+single-cell+transcriptomes+compared+in+matched+cortical+cell+types.">Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.</a> PloS One. 13 (12), e0209648 (2018).</li><li>Roh, H. C., 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=Simultaneous+Transcriptional+and+Epigenomic+Profiling+from+Specific+Cell+Types+within+Heterogeneous+Tissues+In+Vivo.">Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo.</a> Cell Reports. 18 (4), 1048-1061 (2017).</li><li>Aune, U. L., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+differentiation+of+stromal+vascular+cells+to+beige/brite+cells.">Isolation and differentiation of stromal vascular cells to beige/brite cells.</a> Journal of Visualized Experiments. (73), e50191 (2013).</li><li>Zheng, G. X. 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=Massively+parallel+digital+transcriptional+profiling+of+single+cells.">Massively parallel digital transcriptional profiling of single cells.</a> Nature Communications. 8, 14049 (2017).</li><li>Pollen, A. 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=Low-coverage+single-cell+mRNA+sequencing+reveals+cellular+heterogeneity+and+activated+signaling+pathways+in+developing+cerebral+cortex.">Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.</a> Nature Biotechnology. 32 (10), 1053-1058 (2014).</li><li>Hayashi, 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=Single-cell+full-length+total+RNA+sequencing+uncovers+dynamics+of+recursive+splicing+and+enhancer+RNAs.">Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.</a> Nature Communications. 9 (1), 619 (2018).</li><li>Satpathy, A. 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=Massively+parallel+single-cell+chromatin+landscapes+of+human+immune+cell+development+and+intratumoral+T+cell+exhaustion.">Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion.</a> Nature Biotechnology. 37 (8), 925-936 (2019).</li><li>Stoeckius, 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=Simultaneous+epitope+and+transcriptome+measurement+in+single+cells.">Simultaneous epitope and transcriptome measurement in single cells.</a> Nature Methods. 14 (9), 865-868 (2017).</li><li>Peterson, V. 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The authors declare that they have no competing financial interests.

A straightforward and robust method to isolate single nuclei and study adipose tissue heterogeneity is presented. Compared to whole tissue RNA sequencing, this workflow offers an unbiased view of cellular heterogeneity and population-specific markers. This is significant and innovative for the advancement of adipocyte biology, molecular metabolism, and obesity research.
This protocol is particularly optimized for downstream application of snRNA-seq. The &#34;cleanup&#34; step to achieve isolat...

Studies have shown that brown adipose tissue (BAT) has a remarkable capacity to dissipate energy. Two types of thermogenic adipocytes with distinct developmental features exist in both rodents and humans: beige adipocytes and classical brown adipocytes. While classical brown adipocytes are located mostly in interscapular BAT depots, beige adipocytes sporadically emerge in white adipose tissue (WAT) in response to certain physiological cues, such as chronic cold exposure, a process referred to as &#34;browning&#34; or &#34;beiging&#34;. Through the use of advanced imaging, it is now clear that adult humans have substantial depots of UCP1+ BAT, especially in the supraclavicular region1,2,3,4. The amount of adult human BAT inversely correlates with adiposity and can be increased by external cues, such as chronic cold exposure5,6 or &#946;3-adrenergic receptor agonist7. BAT-mediated energy expenditure may offer a viable approach to combat obesity.
Until recently, thermogenic adipocytes have been considered a homogeneous population. However, studies have revealed the existence of multiple subtypes or subpopulations that are distinct in developmental origin, substrate usage, and transcriptome8,9,10. For instance, a type of beige adipocyte that preferentially uses glucose for thermogenesis, the g-beige adipocyte, was recently described10. The incomplete understanding of cell types in brown and beige adipose tissue and the lack of specific markers constitute a critical barrier to studying their biological functions.
Traditional methods for isolating subpopulations of cells are based on expression of only a few known marker genes. Recent advances in single-cell genomics enables the use of global gene expression data of single cells to provide an unbiased estimate of the number of subpopulations in a tissue. The ultimate goal of this protocol is to determine all adipose tissue subtypes under various thermogenic stimuli at a single-cell resolution. In contrast to other tissues and cell types, determining cellular subtypes of adipose tissue is challenging due to the fragility of lipid-filled adipocytes. This paper introduces a robust protocol to isolate single nuclei from adipose tissue for downstream application to snRNA sequencing. Importantly, recent literature comparing well-matched single-nuclei RNA sequencing (snRNA-seq) and single-cell RNA sequencing (scRNA-seq) datasets revealed that snRNA-seq is comparable to scRNA-seq in cell type detection, and superior in cellular coverage for a complex tissue like the brain11. This protocol combines a density gradient centrifugation method optimized for adipose tissues by Rosen et al.12 with a nuclei &#34;cleanup&#34; step with a MoFlo XDP High Speed Sorter. As seen in the representative results, an analysis of 7,500 single nuclei from mouse interscapular brown adipose tissue identified multiple cell types within seemingly homogeneous brown adipocytes. Overall, this simple and robust protocol can be applied to study tissue-level organization of adipocytes and adipose-resident cells, identification of subtype-specific marker genes, and development phenotyping of adipose-selective knockout/transgenic mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>PBS with EDTA; sterile-filtered</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1595</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell filter 100 &#956;m</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell filter 40&#956;m</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics (30 &#956;m)</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217-3</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td>Stackable filters</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFloXDP Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>ML99030</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Protector RNase Inhibitor</td>
			<td>Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S5-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Unsorted adipocyte nuclei contain debris and doublets that create noise and high background in downstream single-cell RNA sequencing. The representative FACS gate strategy is shown in Figure 1. The nuclei were first selected based on forward scatter (FSC) and side scatter (SSC) (A), then, only singlets were selected based on the combination of width and heights of SSC (B). Finally, only DAPI-positive events were selected and ...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications at JoVE.com

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

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 ...

isolation-adipose-tissue-nuclei-for-single-cell-genomic

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9.7K Views.  Albert Einstein College of Medicine. This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Video: Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...