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Method Article

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

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DOI:

10.3791/65328

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May 12th, 2023

 ,  ,  ,  ,  , 
1Center for Cardiovascular Disease & Nutrition, U1263, Aix-Marseille University, 2Marseille Medical Genetics, U1251, Aix-Marseille University

In This Article

Summary

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.

Abstract

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.

Introduction

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. 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.

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Protocol

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 (Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche; Authorization Apafis N°33927-2021111715507212).

1. Setting up the timed mating prior to dissection

  1. 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.
  2. 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.

2. Tissue preparation and cell isolation

  1. 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.
  2. Open the abdomen and peritoneum with scissors. Visualize the uterine horns, and use forceps to excise both horns.
  3. 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.
  4. Under the stereomicroscope set at 5x magnification and using forceps, remove the endometrial tissue, placenta, and yolk sac from each embryo.
  5. 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.
  6. Pool together the heart regions dissected from five mouse embryos in a 1.5 mL microcentrifuge tube. Prechill swinging bucket centrifuges to 4 °C.
  7. 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.
  8. 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).
  9. For tissue digestion, add 50 µL of 0.25% trypsin/EDTA for 10 min at 37 °C. Apply gentle mechanical dissociation after 5 min by up and down gestures using a wide-orifice filter tip.
  10. Inactivate trypsin digestion activity by adding 350 µL of complete medium to the dissociated cells. Pass the resuspended cells through a 40 µm cell strainer.

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.

  1. Perform a trypan blue assay for cell counting, as described below.
    1. Vortex 0.4% trypan blue staining solution, spin for 10 s at room temperature at 2,000 x g, and transfer 10 µL into a microtube.
    2. Using a pipette, mix the cell suspension, and add 10 µL of suspension to the already aliquoted 10 µL of trypan blue solution. Mix carefully 10 times with a pipette.
    3. Transfer 10 µL of the stained cells into a counting slide. Place the slide into the counter to automatically calculate the cell concentration and viability.
  2. 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's instructions were followed for the appropriate preparation and usage.
    1. Prepare a 10 µM EthD-1 solution by adding 5 µL 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.
    2. Transfer 2.5 µL of the supplied 4 mM calcein AM stock solution to the already prepared EthD-1 solution. Vortex for 5 s to ensure complete mixing.
    3. Add 10 µL of the resulting 10 µM EthD-1 and 10 µM calcein AM working solution to 10 µL of cell suspension.
      NOTE: Calcein is highly susceptible to hydrolysis in aqueous solutions, so use this working solution within 1 day.
    4. Transfer 10 µL 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).

4. Cell freezing

  1. Centrifuge the cell suspension at 500 x g for 5 min at 4 °C. Carefully remove the supernatant without touching the bottom of the tube, and resuspend the pellet in 1 mL of chilled freezing medium.
  2. Transfer the cell suspension into a pre-cooled cryovial, and place the cryovial in a pre-cooled freezing container.
  3. Place the container at −80 ◦C for 4 h to 8 h. Transfer the cryovial to liquid nitrogen for downstream single-nucleus RNA and ATAC sequencing experiments.
    ​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 −150 °C all times to prevent the formation of ice crystals.

5. Cell thawing and removal of dead cells

  1. Place the cryovials in a 37 °C water bath for 1-2 min. Remove it when a tiny crystal remains in the cryovial.
  2. Add 1 mL of pre-warmed (37 °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.
  3. Pass the entire cell suspension over a 30 µm strainer. Rinse the strainer with 1-2 mL of warm complete medium, and collect the flowthrough in the same conical tube.
  4. 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.
  5. Centrifuge at 300 x g for 5 min, and remove the supernatant without disrupting the cell pellet.
  6. Add 100 µL 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.
  7. In the meantime, rinse the magnetic separation column (capacity: 1 x 107 to 2 x 108 cells) with 500 µL of 1x binding buffer.
  8. Once the incubation is completed, dilute the cell suspension containing the microbeads with 500 µL of 1x binding buffer.
  9. Apply the cell suspension (0.6 µL) 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.
  10. 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.
  11. Centrifuge at 300 x g for 5 min. Remove the supernatant without disrupting the cell pellet.
  12. 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.
  13. Centrifuge at 300 x g for 5 min. Remove the supernatant without disrupting the cell pellet.
  14. Add 1 mL of PBS-BSA to resuspend the pellet, and repeat the washing step for a total of two washes.
  15. Resuspend the cells in 100 µL of PBS-BSA solution. Mix gently by pipetting 10 times.
  16. 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 ≥100,000 cells. See Figure 2 for representative results.
    ​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.

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.

  1. Clean the working place and materials with 70% ethanol and RNase removing solution.
  2. Prechill swinging bucket centrifuges to 4 °C. Freshly prepare lysis buffer, lysis dilution buffer, 0.1x lysis buffer, and wash buffer (see Table 1). Maintain the buffers on ice.
  3. Centrifuge the cell suspension at 500 x g for 5min at 4 °C in a swinging bucket centrifuge. Gently remove all the supernatant without touching the bottom of the tube to avoid dislodging the cell pellet.
  4. Add 100 µL of chilled 0.1x lysis buffer. Gently mix by pipetting 10 times. Incubate for 5 min on ice.
  5. Add 1 mL of chilled wash buffer, and gently mix by pipetting five times. Centrifuge at 500 x g for 5 min at 4 °C. Remove the supernatant without disrupting the nuclei pellet.
  6. 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.

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's user guide. See an example of this calculation in Supplementary Protocol 1.

  1. 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.
  2. Determine the actual nuclei concentration. Mix 2 µL of nuclei stock solution with 8 µL of diluted nuclei buffer and add the mix to the pre-aliquoted 10 µL 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.
  3. Calculate the volume of nuclei stock and the volume of diluted nuclei buffer to obtain a total volume of 5 µL at the recommended concentration for the transposition reaction (refer to the manufacturer's user guide). Proceed immediately to the transposition reaction according to the user guide17.
    ​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.

8. Quality analysis of the snRNA-seq and snATAC-seq libraries

  1. 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.

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Results

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...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2100 Bioanalyzer InstrumentAgilentNo catalog number
5M Sodium chloride (NaCl)Sigma59222C-500ML 
BSA 10% SigmaA1595-50ML
Chromium Next GEM Chip J Single Cell Kit, 16 rxns10X Genomics1000230
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 4 rxns (including Nuclei Buffer 20X)10X Genomics1000285
Countess cell counting chamber slidesInvitrogenC10283
Countess III FLThermofisherNo catalog number
Digitonin (5%)ThermofisherBN2006
DMSOSigmaD2650-5x5ML
DNA LoBind Tubes Eppendorf22431021
D-PBSThermofisher14190094Sterile and RNase-free
Dual Index Kit TT Set A 96 rxns10X Genomics1000215
Falcon 15 mL Conical Centrifuge Tubes Fisher Scientific 352096
Falcon 50 mL Conical Centrifuge Tubes Fisher Scientific 10788561
HI-FBSThermofisherA3840001Heat inactivated
High sensitivity DNA kitAgilent5067-4626
Igepal CA-630SigmaI8896-50ML
LIVE/DEAD Viability/Cytotoxicity KitThermofisherL3224
MACS Dead Cell Removal kit: Dead Cell Romoval MicroBeads, Binding Buffer 20XMiltenyi Biotec130-090-101
MACS SmartStrainers (30 µm)Miltenyi Biotec130-098-458
Magnesium chloride (MgCl2)SigmaM1028-100ML
Milieu McCoy 5A Thermofisher16600082
MS ColumnsMiltenyi Biotec130-042-201
NovaSeq 6000 S2IlluminaNo catalog number
Penicillin Streptomycin (Pen/Strep)Thermofisher15070063
PluriStrainer Mini 40µmPluriSelectV-PM15-2021-12
Rock inhibitorEnzo Life SciencesALX-270-333-M005
Single Index Kit N Set A, 96 rxn10X Genomics1000212
Standard 90mm Petri dish SterilinThermofisher101R20
Sterile double-distilled waterThermofisherR0582
Trizma Hydrochloride solution (HCl)SigmaT2194-100ML 
Trypan Blue stain (0.4%)InvitrogenT10282
Trypsin 0.05% - EDTA 1XThermofisher25300054
Tween20SigmaP9416-50ML 
Wide orifice filtered pipette tips 200 μlLabcon1152-965-008-9
ZEISS SteREO Discovery.V8ZEISSNo catalog number

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Single Nucleus RNA SeqSingle Nucleus ATAC SeqDual Omics ProfilingFrozen Tissue ProtocolCell Viability AssessmentMagnetic SeparationChromatin AccessibilityHeart Development
Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution
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