This research was supported by ERA-CVD-2019 and ANR-JCJC-2020 to SS. We thank the Genomics and Bioinformatics facility (GBiM) from the U 1251/Marseille Medical Genetics lab and the anonymous reviewers for providing valuable comments.

The animal procedure adopted in this study was approved by the animal ethics committees of the Aix-Marseille University (C2EA-14) and was carried out according to protocols approved by the appointed national ethical committee for animal experimentation (Minist&#232;re de l&#39;Education Nationale, de l&#39;Enseignement Sup&#233;rieur et de la Recherche; Authorization Apafis N&#176;33927-2021111715507212).
1. Setting up the timed mating prior to dissection
<ol>
	<li>To generate mouse embryos, perform a timed mating between adult mice 9.5 days prior to heart region isolation. In this procedure, wild-type C57BL/6J mice aged 2-6 months were used, and timed mating was performed overnight.</li>
	<li>The next morning, check if the female mice have a vaginal plug. Consider the day the plug is identified as embryonic day (E) 0.5.</li>
</ol>
2. Tissue preparation and cell isolation
<ol>
	<li>Euthanize 2-6 months old pregnant female C57BL/6J at day 9.5 post-conception via CO2 with an increasing concentration followed by cervical dislocation. Clean the lower part of the abdomen with 70% ethanol. 
	NOTE: The use of anesthetics before cervical dislocation is not recommended as this would expose the embryos to environmental changes that could impact subsequent transcriptomic and epigenomic studies.</li>
	<li>Open the abdomen and peritoneum with scissors. Visualize the uterine horns, and use forceps to excise both horns.</li>
	<li>Place the horns in a Petri dish containing cold complete medium with 1% FBS. While no specific volume is required, care must be taken to ensure that the embryos are completely immersed in the medium. An approximate volume of 10 mL per 10 cm Petri dish is appropriate.</li>
	<li>Under the stereomicroscope set at 5x magnification and using forceps, remove the endometrial tissue, placenta, and yolk sac from each embryo.</li>
	<li>Place the embryos in a Petri dish with cold complete medium with 1% FBS. Dissect the embryonic heart region, as described on the schematic embryo illustrated in Figure 1, in cold complete medium.</li>
	<li>Pool together the heart regions dissected from five mouse embryos in a 1.5 mL microcentrifuge tube. Prechill swinging bucket centrifuges to 4 &#176;C.</li>
	<li>Centrifuge at 300x g for 1 min at RT in a swinging bucket centrifuge. Remove the supernatant, and wash the tissues by adding 1 mL of tissue culture-grade PBS.</li>
	<li>Centrifuge at 300 x g for 1 min at RT. Remove the supernatant, and repeat the washing steps for a total of two washes. Repeat the wash twice, replacing the PBS with 0.05% trypsin/EDTA (1x).</li>
	<li>For tissue digestion, add 50 &#181;L of 0.25% trypsin/EDTA for 10 min at 37 &#176;C. Apply gentle mechanical dissociation after 5 min by up and down gestures using a wide-orifice filter tip.</li>
	<li>Inactivate trypsin digestion activity by adding 350 &#181;L of complete medium to the dissociated cells. Pass the resuspended cells through a 40 &#181;m cell strainer.</li>
</ol>
3. Cell counting and viability assessment
NOTE: The cell count and viability are critical parameters for the success of single-cell experiments. The snATAC-seq approach is sensitive to minor variations in cell number. Too few cells lead to over-digestion of the chromatin, which results in a higher number of reads and the mapping of inaccessible chromatin regions (noise). Similarly, having too many cells generates high-molecular-weight fragments that are hard to sequence. For the accurate determination of the cell and nuclei concentrations, the cell count and viability were assessed by two different methods.
<ol>
	<li>Perform a trypan blue assay for cell counting, as described below.
	<ol>
		<li>Vortex 0.4% trypan blue staining solution, spin for 10 s at room temperature at 2,000 x g, and transfer 10 &#181;L into a microtube.</li>
		<li>Using a pipette, mix the cell suspension, and add 10 &#181;L of suspension to the already aliquoted 10 &#181;L of trypan blue solution. Mix carefully 10 times with a pipette.</li>
		<li>Transfer 10 &#181;L of the stained cells into a counting slide. Place the slide into the counter to automatically calculate the cell concentration and viability.</li>
	</ol>
	</li>
	<li>Perform a fluorescence-based assessment of mortality as described below. 
	NOTE: This assay is based on the use of fluorescent dyes (ethidium homodimer-1, EthD-1, and calcein AM) to evaluate the cell viability. A commercially available kit was used, and the provider&#39;s instructions were followed for the appropriate preparation and usage.
	<ol>
		<li>Prepare a 10 &#181;M EthD-1 solution by adding 5 &#181;L of the supplied 2 mM EthD-1 stock solution to 1 mL of sterile tissue-culture grade D-PBS, and vortex for 5 s to ensure complete mixing.</li>
		<li>Transfer 2.5 &#181;L of the supplied 4 mM calcein AM stock solution to the already prepared EthD-1 solution. Vortex for 5 s to ensure complete mixing.</li>
		<li>Add 10 &#181;L of the resulting 10 &#181;M EthD-1 and 10 &#181;M calcein AM working solution to 10 &#181;L of cell suspension. 
		NOTE: Calcein is highly susceptible to hydrolysis in aqueous solutions, so use this working solution within 1 day.</li>
		<li>Transfer 10 &#181;L of the stained cells onto a counting slide. Insert the slide into the automated fluorescent cell counter. Count the live cells using a GFP filter and the dead cells with an RFP filter. 
		NOTE: The two counting methods gave similar cell counts and viability, and the total number of freshly dissociated cells was estimated to average around 20,000 cells per embryonic heart region (0.06 mm3).</li>
	</ol>
	</li>
</ol>
4. Cell freezing
<ol>
	<li>Centrifuge the cell suspension at 500 x g for 5 min at 4 &#176;C. Carefully remove the supernatant without touching the bottom of the tube, and resuspend the pellet in 1 mL of chilled freezing medium.</li>
	<li>Transfer the cell suspension into a pre-cooled cryovial, and place the cryovial in a pre-cooled freezing container.</li>
	<li>Place the container at &#8722;80 &#9702;C for 4 h to 8 h. Transfer the cryovial to liquid nitrogen for downstream single-nucleus RNA and ATAC sequencing experiments. 
	&#8203;NOTE: The cryovial should be transported on dry ice prior to usage. In our experience, the cells may be stored in liquid nitrogen for at least 6 months with no loss of cell viability. The storage temperature should be kept below &#8722;150 &#176;C all times to prevent the formation of ice crystals.</li>
</ol>
5. Cell thawing and removal of dead cells
<ol>
	<li>Place the cryovials in a 37 &#176;C water bath for 1-2 min. Remove it when a tiny crystal remains in the cryovial.</li>
	<li>Add 1 mL of pre-warmed (37 &#176;C) complete medium. Mix with a pipette three times. Transfer the suspension to a 15 mL conical tube containing 10 mL of warm complete medium.</li>
	<li>Pass the entire cell suspension over a 30 &#181;m strainer. Rinse the strainer with 1-2 mL of warm complete medium, and collect the flowthrough in the same conical tube.</li>
	<li>Centrifuge at 300 x g for 5 min, and remove supernatant. Wash one time with PBS, and transfer the cell suspension into a 1.5 mL microtube.</li>
	<li>Centrifuge at 300 x g for 5 min, and remove the supernatant without disrupting the cell pellet.</li>
	<li>Add 100 &#181;L of the provided magnetic microbeads to the pelleted cells, and homogenize five times using a wide-bore pipette tip. Incubate for 15 min at RT.</li>
	<li>In the meantime, rinse the magnetic separation column (capacity: 1 x 107 to 2 x 108 cells) with 500 &#181;L of 1x binding buffer.</li>
	<li>Once the incubation is completed, dilute the cell suspension containing the microbeads with 500 &#181;L of 1x binding buffer.</li>
	<li>Apply the cell suspension (0.6&#160;&#181;L) to the prepared magnetic separation column. The flow-through is the negatively selected live cell fraction, and the magnetically retained fraction is the positively selected dead cells.</li>
	<li>Collect the live cell effluent in a 15 mL centrifuge tube. Rinse the 1.5 mL microtube that contained the cell suspension and the column with 2 mL of 1x binding buffer, and collect the effluent in the same 15 mL tube.</li>
	<li>Centrifuge at 300 x g for 5 min. Remove the supernatant without disrupting the cell pellet.</li>
	<li>Add 1 mL of the PBS-BSA solution, and mix gently by pipetting five times using a wide-orifice pipette tip. Transfer the cell suspension into a 1.5 mL microtube.</li>
	<li>Centrifuge at 300 x g for 5 min. Remove the supernatant without disrupting the cell pellet.</li>
	<li>Add 1 mL of PBS-BSA to resuspend the pellet, and repeat the washing step for a total of two washes.</li>
	<li>Resuspend the cells in 100 &#181;L of PBS-BSA solution. Mix gently by pipetting 10 times.</li>
	<li>Determine the concentration and viability of the cells using the previously described methods (step 3.1 and step 3.2). To proceed to the next step, ensure a cell count of &#8805;100,000 cells. See Figure 2 for representative results. 
	&#8203;NOTE: Cryopreservation results in a loss of approximately 40% of the initial starting material of live cells. Depending on the starting number of cells, bead separation on a magnetic column results in high cell viability but divides the total number of cells by two-fold to five-fold. The utilization of a column-based magnetic kit to remove the dead cells is advised only when sufficient starting material is available.</li>
</ol>
6. Nuclei isolation
NOTE: snATAC and snRNA-seq combined are performed with a suspension of clean and intact nuclei. The optimization of the lysis conditions (lysis time and NP40 concentration) for the used cell type is recommended. The optimal lysis timepoint and detergent concentration are those that result in the maximum number of cells being lysed without disrupting the nuclear morphology. The loss of cells/nuclei can be reduced by using a swinging bucket centrifuge instead of a fixed-angle centrifuge. To minimize nuclei retention on plastics, it is recommended to use low-retention pipette tips and centrifuge tubes. This can increase the nuclei recovery. The coating of the pipette tips and centrifuge tubes with 5% BSA is a less expensive but more time-consuming alternative.
<ol>
	<li>Clean the working place and materials with 70% ethanol and RNase removing solution.</li>
	<li>Prechill swinging bucket centrifuges to 4 &#176;C. Freshly prepare lysis buffer, lysis dilution buffer, 0.1x lysis buffer, and wash buffer (see Table 1). Maintain the buffers on ice.</li>
	<li>Centrifuge the cell suspension at 500 x g for 5min at 4 &#176;C in a swinging bucket centrifuge. Gently remove all the supernatant without touching the bottom of the tube to avoid dislodging the cell pellet.</li>
	<li>Add 100 &#181;L of chilled 0.1x lysis buffer. Gently mix by pipetting 10 times. Incubate for 5 min on ice.</li>
	<li>Add 1 mL of chilled wash buffer, and gently mix by pipetting five times. Centrifuge at 500 x g for 5 min at 4 &#176;C. Remove the supernatant without disrupting the nuclei pellet.</li>
	<li>Repeat the washes with chilled wash buffer for a total of three washes. Prepare the diluted nuclei buffer. After resuspension, keep the nuclei stock solution on ice.</li>
</ol>
7. Quality and quantity assessments of the isolated nuclei
NOTE: Based on the initial cell count and estimating approximately 50% nuclei loss during cell lysis, resuspend the appropriate number of cells in cold diluted nuclei buffer. The calculation of the resuspension volume with chilled diluted nuclei buffer is based on the targeted nuclei recovery and the corresponding nuclei stock concentrations recommended by the manufacturer&#39;s user guide. See an example of this calculation in Supplementary Protocol 1.
<ol>
	<li>Based on the initial cell count and estimating approximately 50% nuclei loss during cell lysis, resuspend the appropriate number of cells in cold diluted nuclei buffer.</li>
	<li>Determine the actual nuclei concentration. Mix 2 &#181;L of nuclei stock solution with 8 &#181;L of diluted nuclei buffer and add the mix to the pre-aliquoted 10 &#181;L of trypan blue or EthD-1/calceinAM working solution. Count the nuclei using an automated cell counter. Less than 5% of the cells should be viable. See Figure 3 for representative results.</li>
	<li>Calculate the volume of nuclei stock and the volume of diluted nuclei buffer to obtain a total volume of 5 &#181;L at the recommended concentration for the transposition reaction (refer to the manufacturer&#39;s user guide). Proceed immediately to the transposition reaction according to the user guide17. 
	&#8203;NOTE: All the steps from the transposition reaction to the construction of the ATAC and GEX libraries were performed according to the user guide of the kit used for snRNA-seq and snATAC-seq combined17.</li>
</ol>
8. Quality analysis of the snRNA-seq and snATAC-seq libraries
<ol>
	<li>Before proceeding to next-generation sequencing, validate the quality and size distribution of the final snATAC-seq and gene expression GEX libraries. Assess the quality and quantity of sequences identified in the libraries using fragment analysis systems. See Figure 4 for representative quality assessment results. 
	NOTE: The final trace of the snATAC-seq libraries should show the periodicity of nucleosome winding, and the sizes should range from 200 bp to several Kbp, whereas the sizes in the gene expression library should range from 300 bp to 600 bp.</li>
</ol>

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

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<li>Stefanovic, S., Etchevers, H. C., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Outflow+tract+formation-embryonic+origins+of+conotruncal+congenital+heart+disease%5BTitle%5D)+AND+%22Journal+of+Cardiovascular+Development+and+Disease%22%5BJournal%5D)">Outflow tract formation-embryonic origins of conotruncal congenital heart disease.</a> Journal of Cardiovascular Development and Disease. 8 (4), 42(2021).</li>
<li>Hao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+analysis+of+multimodal+single-cell+data%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Integrated analysis of multimodal single-cell data.</a> Cell. 184 (13), 3573-3587 (2021).</li>
<li>Bai, H., Lin, M., Meng, Y., Bai, H., Cai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+CUT%26RUN+method+for+regulation+network+reconstruction+of+low+abundance+transcription+factor%5BTitle%5D)+AND+%22Cellular+Signalling%22%5BJournal%5D)">An improved CUT&RUN method for regulation network reconstruction of low abundance transcription factor.</a> Cellular Signalling. 96, 110361(2022).</li>
</ol>

The authors have nothing to disclose.

The analysis of the cellular composition of the developing heart by combined snRNA-seq and snATAC-seq studies provides a deeper understanding of the origin of congenital heart disease26. Several research laboratories have studied the effects of cardiac tissue cryopreservation on snRNA-seq27. Conducting snRNA-seq and snATAC-seq using fresh micro-dissected tissue from mouse models of human disease can be logistically challenging when comparing different experimental condition...

Among birth defects, congenital heart defects (CHDs) are the most common, occurring in about 1% of live births each year1,2. Genetic mutations are identified in only a minority of cases, implying that other causes, such as abnormalities in gene regulation, are involved in the etiology of CHD2,3. Cardiac development is a complex process of diverse and interacting cell types, making the identification of causal noncoding mutations and their effects on gene regulation challenging. Organogenesis of the heart begins with cellular progenitors that give rise to different subtypes of cardiac cells, including myocardial, fibroblast, epicardial, and endocardial cells4,5.&#160;Single-cell genomics is emerging as a key method for studying heart development and assessing the impact of cellular heterogeneity in health and disease6. The development of multi-omics methods for the simultaneous measurement of different parameters and the expansion of computational pipelines has facilitated the discovery of cell types and subtypes in the normal and diseased heart6. This article describes a reliable single-nucleus isolation protocol for frozen cardiac progenitor cells obtained from mouse embryos that is compatible with downstream snRNA-seq and snATAC-seq (as well as snRNA-seq and snATAC-seq combined)7,8,9.
ATAC-seq is a robust method that allows the identification of regulatory open chromatin regions and the positioning of nucleosomes10,11. This information is used to draw conclusions about the location, identity, and activity of transcription factors. The activity of chromatin factors, including remodelers, as well as the transcriptional activity of RNA polymerase, can, thus, be analyzed since the method is highly sensitive for measuring quantitative changes in chromatin structure1,2. Thus, ATAC-seq provides a robust and impartial approach to uncovering the mechanisms controlling transcriptional regulation in a specific cell type. ATAC-seq protocols have also been validated to measure chromatin accessibility in single cells, revealing variability in the chromatin architecture within cell populations10,12,13.
Although there have been notable advances in the field of single cells in recent years, the main difficulty is the processing of the fresh samples needed to perform these experiments14. To circumvent this difficulty, various tests have been carried out with the aim of conducting analyses such as snRNA-seq and snATAC-seq with frozen cardiac tissue or cells15,16.
Several platforms have been used to analyze single-cell genomics data17. The widely used platforms for single-cell gene expression and ATAC profiling are platforms for multiple microfluidic droplet encapsulation17. As these platforms use microfluidic chambers, debris or aggregates can clog the system, resulting in non-usable data. Thus, the success of single-cell studies depends on the accurate isolation of individual cells/nuclei.
The protocol presented here uses a similar approach to recent studies using snRNA-seq and snATAC-seq to understand congenital heart defects18,19,20,21,22,23. This procedure utilizes the enzymatic dissociation of freshly microdissected cardiac tissue followed by the cryopreservation of mouse cardiac progenitor cells. After thawing, the viable cells are purified and processed for nuclear isolation. In this work, this protocol was successfully used to obtain snRNA-seq and snATAC-seq data from the same nuclear preparation of mouse cardiac progenitor cells.

<table><tbody><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent</td><td></td><td>No catalog number</td></tr><tr><td>5M Sodium chloride (NaCl)</td><td>Sigma</td><td>59222C-500ML&amp;nbsp;</td><td></td></tr><tr><td>BSA 10%&amp;nbsp;</td><td>Sigma</td><td>A1595-50ML</td><td></td></tr><tr><td>Chromium Next GEM Chip J Single Cell Kit, 16 rxns</td><td>10X Genomics</td><td>1000230</td><td></td></tr><tr><td>Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 4 rxns (including Nuclei Buffer 20X)</td><td>10X Genomics</td><td>1000285</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>Countess III FL</td><td>Thermofisher</td><td></td><td>No catalog number</td></tr><tr><td>Digitonin (5%)</td><td>Thermofisher</td><td>BN2006</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-5x5ML</td><td></td></tr><tr><td>DNA LoBind Tubes&amp;nbsp;</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>D-PBS</td><td>Thermofisher</td><td>14190094</td><td>Sterile and RNase-free</td></tr><tr><td>Dual Index Kit TT Set A 96 rxns</td><td>10X Genomics</td><td>1000215</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>352096</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>10788561</td><td></td></tr><tr><td>HI-FBS</td><td>Thermofisher</td><td>A3840001</td><td>Heat inactivated</td></tr><tr><td>High sensitivity DNA kit</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Igepal CA-630</td><td>Sigma</td><td>I8896-50ML</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit</td><td>Thermofisher</td><td>L3224</td><td></td></tr><tr><td>MACS Dead Cell Removal kit: Dead Cell Romoval MicroBeads, Binding Buffer 20X</td><td>Miltenyi Biotec</td><td>130-090-101</td><td></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>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>M1028-100ML</td><td></td></tr><tr><td>Milieu McCoy 5A&amp;nbsp;</td><td>Thermofisher</td><td>16600082</td><td></td></tr><tr><td>MS Columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>NovaSeq 6000 S2</td><td>Illumina</td><td></td><td>No catalog number</td></tr><tr><td>Penicillin Streptomycin (Pen/Strep)</td><td>Thermofisher</td><td>15070063</td><td></td></tr><tr><td>PluriStrainer Mini 40&amp;micro;m</td><td>PluriSelect</td><td>V-PM15-2021-12</td><td></td></tr><tr><td>Rock inhibitor</td><td>Enzo Life Sciences</td><td>ALX-270-333-M005</td><td></td></tr><tr><td>Single Index Kit N Set A, 96 rxn</td><td>10X Genomics</td><td>1000212</td><td></td></tr><tr><td>Standard 90mm Petri dish Sterilin</td><td>Thermofisher</td><td>101R20</td><td></td></tr><tr><td>Sterile double-distilled water</td><td>Thermofisher</td><td>R0582</td><td></td></tr><tr><td>Trizma Hydrochloride solution (HCl)</td><td>Sigma</td><td>T2194-100ML&amp;nbsp;</td><td></td></tr><tr><td>Trypan Blue stain (0.4%)</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>Trypsin 0.05% - EDTA 1X</td><td>Thermofisher</td><td>25300054</td><td></td></tr><tr><td>Tween20</td><td>Sigma</td><td>P9416-50ML&amp;nbsp;</td><td></td></tr><tr><td>Wide orifice filtered pipette tips 200 &amp;mu;l</td><td>Labcon</td><td>1152-965-008-9</td><td></td></tr><tr><td>ZEISS SteREO Discovery.V8</td><td>ZEISS</td><td></td><td>No catalog number</td></tr></tbody></table>

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.

Compared to the preparation of single-cell suspensions for single-cell approaches, the preparation of single-nuclei suspensions is much more challenging and requires a higher degree of resolution and processing. The key factor for successful combined snRNA-seq and snATAC-seq is a clean and intact nuclei suspension. The protocol for efficient nuclei isolation must be adapted to each tissue type and condition (fresh or frozen). Here, an optimized protocol is described for the isolation of nuclei from frozen mouse embryonic...

Watch this Scientific Journal Video about Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution at JoVE.com

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.

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 ...

nuclei-isolation-from-mouse-cardiac-progenitor-cells-for-epigenome

Research

JoVE Journal

Developmental Biology

3.0K Views.  Aix-Marseille University. 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. 

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

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

High-Quality Nuclei from iPSC-Derived Hematopoietic Cells for Multiomics Studies

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Author Spotlight: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Biology

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Neuroscience

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

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 ...