Ana Jeemin Choi was supported by a stipend from the Pasteur - Paris University (PPU) International Ph.D. Program.

High-Quality Sample Preparation for Multiome 10X Genomics Analysis

<ol>
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The authors have nothing to disclose.

Preparation of high-quality cell or nuclei suspension is of crucial importance for the success of single-cell or single-nuclei RNA-Seq and single-cell multi-omic analyses29,30,31. Here, we have described protocols for sample preparation and nuclei isolation for multiome assays from two types of tissue: brain and bone marrow.
The brain protocol described in this paper allows the recovery of high-quality nuclei from fresh-frozen brain tissue. It includes the following steps: frozen tissue disruption, isolation of nuclei, purification of nuclei, and quality control of the prepared material. The brain tissue is composed of many different cell types, and the procedure of tissue dissociation and nuclei isolation should preserve the proportions of cell populations as present in the initial tissue. Here, the lysis buffer composition and incubation time were optimized to enable complete and gentle lysis of all cell populations that compose the tissue.
The bone marrow HSPCs protocol is somewhat different since it requires one additional step at the beginning of the experiment to isolate the cell population of interest from a heterogeneous cellular suspension. After the collection of fresh tissue, red blood cells are lysed, and the sample is enriched for the cell subset of interest. The targeted cells are lysed, the nuclei are isolated, and the quality of the prepared material is controlled.
10X Genomics provides several protocols validated for nuclei isolation in numerous different tissues32,33. The company also commercializes a nuclei isolation kit with a straightforward pipeline for isolating nuclei from validated tissues34. However, these protocols need additional optimization to tailor the particularities of certain samples. An example is the samples that require working with low cell input. For these samples, the most challenging steps are centrifugations that need to be sufficiently stringent to clean the sample and gentle enough to avoid cell/nuclei loss. With the protocol described here, we have adapted the 10X Genomics Demonstrated Protocol - Nuclei Isolation for Single Cell Multiome ATAC + GEX Sequencing (CG000365 - Rev C)27 to find a fine balance between these two requirements. As demonstrated in the example of the preparation of nuclei from sorted HSPCs, we have improved the nuclei recovery with no impact on the quality of the sample.
An additional challenge is the step of lysis of purified cells for nuclei isolation. Harsher lysis conditions and longer incubation times can damage nuclei and thereby impact the quality of sequencing data. Figure 5 shows representative nuclei imaging from bone marrow samples upon different incubation times with lysis buffer and illustrates how different the state of nuclei could be depending on the cell lysis. In the example of the HSPCs, we have identified 3 min lysis as the condition that results in the highest proportion of healthy-looking, intact nuclei and the lowest proportion of damaged nuclei. The lysis incubation times should be optimized for each new type of sample.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65715/65715fig05.jpg" /> 
Figure 5:&#160;Nuclei quality control by microscopy. Shown are representative brightfield images of isolated nuclei from mouse bone marrow with (A) intact and (B) damaged nuclei. Scale bar 5 &#181;m. Images were taken with an inverted microscope using a 40x ELWD NA 0.60 objective and 1.5x digital zoom. <a href="https://www.jove.com/files/ftp_upload/65715/65715fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Both protocols detailed in this work rely on purifying targeted cells or nuclei by high-throughput FACS instruments. This step is of crucial importance for single-cell/nuclei preparation protocols where rare subsets of cells are to be isolated from heterogeneous suspensions. In these, like in the example shown here for HSPCs sorting, a high-dimensional flow cytometry panel may be required to enable &#34;gating&#34; on the cell population of interest. The sorting is extremely fast and accurate, leading to over 95% purity of the sorted cell subsets. This approach exposes the cellular suspension to a pressure of up to 70 psi and may therefore, be limiting for sorting of fragile cells (e.g., dendritic cells, neutrophils) since it may cause the rupture of their cell membrane. In these cases, alternative solutions should be selected for cellular purification, including magnetic sorting, application of new generation instruments (e.g., CellenOne, Cellenion; MACSQuant Tyto, Miltenyi)35,36, or droplet-based systems (e.g., ODIN, Sensific)37. Nevertheless, the slow sorting speed of these technologies, with cell sorting that lasts for hours instead of minutes, is a strong limiting factor for the application of these approaches in the preparation of viable cells for Multiome and other single-cell applications based on analysis of large cell numbers.
For the purification of nuclei isolated from the tissue, FACS is the method of choice due to its throughput and the purity of the isolated material. Nuclei are not sensitive to pressure, and filtered tissue isolates can easily be purified through the cell sorter. If the laboratory is not equipped with a FACS instrument, other alternatives exist, somewhat less efficient but sufficiently good. Examples include ultracentrifugation or the use of small equipment such as MARS (Applied Cell) that separates particles based on their difference in size, using acoustic waves; CURIOX laminar washer that uses hydrophobic properties of cell/nuclei suspensions; or LEVITAS bio that relies on physical properties of cells (levitation) to separate them from the debris.
Here, we describe protocols to obtain a high number of nuclei and the best purity for the downstream Multiome protocol. FACS sorting and repeated centrifugation steps result in a substantial loss of the initial material. For this reason, in the protocol for nuclei preparation from the brain that we describe here requires sufficiently abundant starting material to result in the collection of at least 500,000 nuclei after the FACS sorting. Alternative protocols should be applied if this criterion cannot be matched. When working with rare cell populations or small tissue sections, the available amount of initial material can be a limiting factor. To address this issue, it is possible to improve nuclei recovery by (a) reducing the volume of lysis, (b) reducing the washing volume, (c) using a single wash with extended centrifugation time to try to improve recovery as indicated in 10X Genomics protocols for low cell input nuclei isolation. For multiomic analysis of low-content material, it is worth considering plate-based applications such as scNMT, SNARE-seq, and Paired-seq38 that require much fewer input samples.
In summary, we have described two robust protocols for the preparation of nuclei from the brain and the bone marrow HSPCs for downstream Multiome analysis. These protocols are applicable in any scientific project that requires high-quality single nuclei suspensions from these two types of tissue, irrespective of the scientific question posed. Our group has been applying the brain nuclei isolation protocol in studies of brain development upon inactivation of various targeted genes and in studies of immune response in the context of neurological diseases. We are using the bone marrow nuclei isolation protocol for deciphering the participation of various hematopoietic subpopulations in the establishment of the immune system.

For many years, single-cell techniques have been the gold standard for the analysis of biological processes. They were initially restricted to single-cell phenotyping through microscopy, flow cytometry, and similar assays. A breakthrough in single-cell analysis came with the development of approaches for single-cell molecular profiling, in particular single-cell RNA sequencing (scRNA-Seq) that enabled characterizing the entire transcriptome of individual cells. Highly powerful, scRNA-Seq generates information about the transcriptional status of a cell in a specific condition and time point. However, it does not provide visibility on the gene regulation that drives transcription, or on the molecular modifications that occur over time. To overcome this limitation, many efforts have been invested in the development of single-cell multi-omics assays that enable the analysis of multiple factors and processes from the same cell1,2,3,4. The first successful measurement of two modalities within single cells came through linking multiplex surface protein expression patterns with the full transcriptome of individual cells in the CITE-Seq approach5. More recent evolutions combine gene expression with chromatin accessibility (Assay for Transposase-Accessible Chromatin using sequencing, ATAC-Seq), thereby simultaneously capturing transcriptomic and epigenomic modalities in the same cells (e.g., sci-CAR)6. The first commercial solutions that allowed associating transcriptomics with cell phenotype or with epigenetic changes of the same cell came from 10X Genomics.
Experiments for single-cell molecular profiling contain the following steps: (1) tissue dissociation or preparation of single-cell suspensions; (2) cell purification and/or nuclei isolation; (3) partitioning and barcoding; (4) library construction and quality control; (5) next-generation sequencing; (6) data analysis. While steps (3)-(6) may significantly vary depending on the technology employed, the initial steps are generally common to all of them. The quality of the prepared cell/nuclei suspension will determine the overall outcome of the experiment. Depending on the type of tissue, obtaining high-quality single-cell/nuclei suspensions may be challenging. The particularities of some tissues, such as the heart, the muscle, the brain, the lung, the intestine, and others, require methods for tissue disruption and nuclei isolation adapted to each type of sample in order to guarantee the production of high-quality nuclei for molecular analysis7,8,9,10. The tissue disruption methods and dissociation protocols can be mechanical, enzymatic (e.g., a mix of collagenases and DNase), or a combination of the two, and can be performed manually or by instruments (e.g., Qiagen DSC-400, gentleMACS).
Single-cell techniques have become a tool of choice for biomedical research. In neurobiology, the cell diversity in the brain and the complexity of their functions require high-resolution and high-throughput analysis for visualization of rare cell populations and for assessing their heterogeneity11,12,13,14. Linking cellular identity and gene regulatory mechanisms of individual cells provides insights into brain development and physiology. Another example is studies of immune response in the context of infectious, autoimmune, or cancer diseases, which strongly rely on single-cell analyses. The heterogeneity of immune cell subsets and the complexity of their activity and interactions with other cell types require single-cell resolution in deciphering the mechanisms underlying immune response. The immune cells originate from the bone marrow, where hematopoietic progenitors are composed of gradually differentiating cells that acquire and lose cell surface markers throughout a stepwise process prior to exiting the bone marrow to home in the periphery. Single-cell analysis allows for minute characterization of cellular developmental stages. It can be achieved through single-cell phenotyping, conventionally performed by multi-parameter flow cytometry. However, single-cell transcriptomic signatures have been shown to reveal a more precise identification of progenitor cell subtypes since these cells are distributed in clusters that fall into one another and can therefore be misidentified when using a coarse cell surface marker approach15. An increasing number of studies uncover the epigenetic modifications that hematopoietic stem and progenitor cells (HSPCs) can acquire from exposure to various agents, leading to a significant impact on the long-term responsiveness of the immune system16,17,18,19. The novel multi-omics technologies enable studying these processes with single-cell resolution.
Many protocols for cell and nuclei isolation have been described for brain11,20,21,22 and bone marrow samples23,24. To minimize bias due to experimental variability, it is necessary to validate optimized single-nuclei preparation protocols for joint single-cell transcriptomic and epigenomic sequencing, thereby ensuring the reproducibility of single-cell multiomic assays.
Here, two robust protocols for nuclei preparation from (1) fresh-frozen brain tissue and (2) fresh bone marrow HSPCs for downstream single-cell Multiome analysis are described (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65715/65715fig01.jpg" /> 
Figure 1: Schematic representation of protocols for nuclei isolation from fresh-frozen brain and bone marrow tissues. <a href="https://www.jove.com/files/ftp_upload/65715/65715fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G x 1 &#189; (1.2 mm x 38 mm) Agani needles</td>
			<td>Terumo</td>
			<td>AN*1838S1</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom FACS tube with cell strainer cap 35 &#181;m</td>
			<td>falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>BD pharmagen</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD117 (c-Kit)</td>
			<td>BioLegend</td>
			<td>105812</td>
			<td>Clone: 2B8</td>
		</tr>
		<tr>
			<td>APC/Cyanine 7 anti-mouse CD16/32 (Fc&#947;R)</td>
			<td>BioLegend</td>
			<td>101328</td>
			<td>Clone: 93</td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td>non-applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva Software v8.0.1</td>
			<td>BD Biosciences</td>
			<td>non-applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160; stock solution 10%</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell staining buffer</td>
			<td>Biolegend</td>
			<td>420201</td>
			<td></td>
		</tr>
		<tr>
			<td>CFI Suprplan Fluor ELWD 40XC ON 0.6</td>
			<td>Nikon&#160;</td>
			<td>non-applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera Prime 95B 25 mm</td>
			<td>Photometrix</td>
			<td>non-applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slide</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass 24 mm x 24 mm 0.13-0.17 mm</td>
			<td>Brand</td>
			<td>BR470819</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonine 5%</td>
			<td>Invitrogen</td>
			<td>BN2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpels&#160;</td>
			<td>Swann-Morton&#160;</td>
			<td>0508</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x) + GlutaMAX-I</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (10x)</td>
			<td>Gibco</td>
			<td>14200-067</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence inverted microscope Nikon Ti2 -E</td>
			<td>Nikon&#160;</td>
			<td>non-applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 0.5 mL</td>
			<td>Eppendorf</td>
			<td>30123603</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR</td>
			<td>83801.290</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD34</td>
			<td>Invitrogen</td>
			<td>11-0341-85</td>
			<td>Clone: RAM34</td>
		</tr>
		<tr>
			<td>Forceps for dissection</td>
			<td>FST</td>
			<td>11152-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>11533387</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogeniser 2 mL</td>
			<td>Bellco glass</td>
			<td>1984-10002</td>
			<td>Pestle &#8220;A&#8221; Large Clearance: .0030-.0050&#8243; and Pestle &#8220;B&#8221; Small Clearance: .0005-.0025&#8243;</td>
		</tr>
		<tr>
			<td>LIVE/DEAD fixable aqua dead cell stain kit</td>
			<td>Invitrogen</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution 1 M</td>
			<td>Sigma aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Fluoromount-G&#160;</td>
			<td>invitrogen</td>
			<td>00-4958-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P40 substitute</td>
			<td>Sigma aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>ThermoFischer</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei buffer 20x&#160;</td>
			<td>10X Genomics</td>
			<td>2000153/2000207</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei isolation kit EZ prep</td>
			<td>Sigma Aldrich</td>
			<td>NUC-101</td>
			<td></td>
		</tr>
		<tr>
			<td>OneComp eBeads compensation beads</td>
			<td>Invitrogen</td>
			<td>01-1111-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue anti-mouse lineage cocktail 
			(including anti-mouse CD3, Ly-6G/Ly-6C, CD11b, CD45R/B220, TER-119)</td>
			<td>BioLegend</td>
			<td>133310</td>
			<td>Clones (in the same order as the antibodies listed): 17A2, RB6-8C5, M1/70, RA3-6B2, Ter-119</td>
		</tr>
		<tr>
			<td>PCR Tube Strips 0.2 mL</td>
			<td>Eppendorf</td>
			<td>951010022</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse Ly-6A/E (Sca-1)</td>
			<td>BioLegend</td>
			<td>122507</td>
			<td>Clone: E13-161.7</td>
		</tr>
		<tr>
			<td>Petri dish 100 mm x 20 mm OPTILUX</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Ply-L-lysine 0.01% sterile-filtered suitable for cell culture</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Printed microscope slides 8 well 6 mm numbered</td>
			<td>Epredia</td>
			<td>ER-301B-CE24</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30108116</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Rnase inhibitor 5000 U</td>
			<td>Takara</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors for dissection</td>
			<td>FST</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 5 M</td>
			<td>Sigma aldrich</td>
			<td>59222C</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, PES, 0.2 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>15206869</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nail polish&#160;</td>
			<td>any</td>
			<td>non-applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride solution pH 7.4</td>
			<td>Sigma aldrich</td>
			<td>T2194</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%</td>
			<td>gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Biorad&#160;</td>
			<td>1662404</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Distilated Water Dnase/Rnase Free</td>
			<td>Invitrogen</td>
			<td>10977-035</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-quality brain and bone marrow nuclei preparation for single nuclei multiome assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

The two protocols described above detail the isolation of nuclei starting from two different types of tissue. The differences and similarities between the two protocols are schematically represented in Figure 1.
Purification of nuclei from mouse brain 
In the protocol described here, we propose a gentle method for nuclei preparation from brain samples. It starts with a mechanical dissociation of the brain tissue in a lysis buffer, followed by washing and strainer filtering steps that remove the remaining tissue from the suspension. The subsequent removal of debris, non-lysed cells, and small particles is achieved by FACS to guarantee that only purified nuclei are loaded for downstream Multiome protocol. Figure 2 shows the profile of nuclei before and after sorting. After the filtering and before nuclei sorting, the sample contains a high quantity of debris, with more than 99% of the &#34;singlets&#34; positive for nuclear stain (7-AAD), indicating optimal cell lysis (Figure 2A). Nuclei are sorted based on the 7-AAD positive gate. A fraction of sorted material is acquired to verify the purity of the prepared nuclei. Figure 2B shows the profile of the brain nuclei after sorting. The nuclei sorting allowed an increase in the nuclei purity from the initial 36% (Figure 2A) to almost 100% (Figure 2B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65715/65715fig02.jpg" /> 
Figure 2: Gating strategy for nuclei sorting and purity test after sorting. Nuclei were stained with 7-AAD and acquired by the cell sorter. (A) Nuclei are first gated based on their size and granularity (FSC-A and SSC-A, respectively). Single particles are then selected based on their FSC-A/FSC-H properties and 7-AAD staining.&#160;(B) After cell sorting, a fraction of the nuclei from the collection tube is tested for purity using the same gating strategy as in A. <a href="https://www.jove.com/files/ftp_upload/65715/65715fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Purification of mouse bone marrow hematopoietic stem cell progenitors (HSPCs) 
Upon isolation from the bone marrow, up to 2 x 105 HSPCs are sorted by FACS, according to the gating strategy shown in Figure 3A. The sorting efficacy and sample purity are assessed (Figure 3B).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65715/65715fig03v2.jpg" /> 
Figure 3: Gating strategy for sorting of bone marrow HSPCs.&#160;(A)&#160;A representative FACS gating strategy for sorting viable LKS+ hematopoietic stem cells and CD34+ myeloid progenitors for nuclei isolation. (B)&#160;Representative FACS plots used for verification of sorted cell population purity. Shown are the proportions of different cell subsets with respect to the parent population. *The two sorted populations. <a href="https://www.jove.com/files/ftp_upload/65715/65715fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The &#34;Low Cell Input Nuclei Isolation&#34; protocol allows nuclei isolation from samples with a maximum of 105 cells. It includes a low number of centrifugation steps, thereby minimizing cell/nuclei loss. We have adjusted the volume of lysis and wash buffers proportionally to the cell input and increased the centrifugation time for maximal nuclei recovery. We have performed a pilot experiment to assess the quantity of recovered nuclei by counting and their quality by flow cytometry and microscopy imaging. Figure 4 shows the HSPCs sample after cell lysis. This protocol generated high-quality nuclei, as observed in Figure 5A, without any debris that could impact the downstream multiome protocol.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65715/65715fig04.jpg" /> 
Figure 4: Purity test sorting of isolated bone marrow HSPC nuclei.&#160;Nuclei were stained with 7-AAD and acquired by the cell sorter. Nuclei were first gated based on their size and granularity (FSC-A and SSC-A, respectively) to assess the purity of the sample. The proportion of nuclei is indicated with respect to the parent population. <a href="https://www.jove.com/files/ftp_upload/65715/65715fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tube number</td>
			<td>Tube name</td>
			<td>Stained entity</td>
			<td>Amount of stained entity</td>
			<td>Antibody/dye (&#181;L)</td>
			<td>Collection Buffer (&#181;L)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Unstained</td>
			<td>Cells</td>
			<td>5,00,000</td>
			<td>N/A</td>
			<td rowspan="7">200</td>
		</tr>
		<tr>
			<td>2</td>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain&#160;</td>
			<td>Cells</td>
			<td>5,00,000</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>3</td>
			<td>APC/Cyanine 7 anti-mouse CD16/32 (Fc&#947;R)</td>
			<td>OneComp eBeads</td>
			<td>15 &#181;L</td>
			<td>1</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Pacific Blue anti-mouse Lineage Cocktail</td>
			<td>OneComp eBeads</td>
			<td>15 &#181;L</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>PE anti-mouse Ly-6A/E (Sca-1)</td>
			<td>OneComp eBeads</td>
			<td>15 &#181;L</td>
			<td>1</td>
		</tr>
		<tr>
			<td>6</td>
			<td>APC anti-mouse CD117 (c-Kit)</td>
			<td>OneComp eBeads</td>
			<td>15 &#181;L</td>
			<td>1</td>
		</tr>
		<tr>
			<td>7</td>
			<td>FITC anti-mouse CD34</td>
			<td>OneComp eBeads</td>
			<td>15 &#181;L</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Single stain controls for compensation settings on the flow cytometer. Indicated are the required single stain controls, the number of cells or beads to be stained, and the antibody quantities.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Master Mix</td>
			<td>Reagent</td>
			<td>Final dilution</td>
			<td>Antibody/dye&#160; (&#181;L)</td>
			<td>Buffer type</td>
			<td>Buffer (&#181;L)</td>
		</tr>
		<tr>
			<td rowspan="2">Mix 1</td>
			<td>APC/Cyanine 7 anti-mouse CD16/32 (Fc&#947;R)</td>
			<td>1/500</td>
			<td>1.2</td>
			<td rowspan="2">DPBS</td>
			<td rowspan="2">300</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain&#160;</td>
			<td>1/250</td>
			<td>2.4</td>
		</tr>
		<tr>
			<td rowspan="4">Mix 2</td>
			<td>Pacific Blue anti-mouse Lineage Cocktail</td>
			<td>1/20</td>
			<td>30</td>
			<td rowspan="4">FACS buffer</td>
			<td rowspan="4">300</td>
		</tr>
		<tr>
			<td>PE anti-mouse Ly-6A/E (Sca-1)</td>
			<td>1/200</td>
			<td>3</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD117 (c-Kit)</td>
			<td>1/200</td>
			<td>3</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD34</td>
			<td>1/50</td>
			<td>12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Total staining volume&#160;</td>
			<td>600</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of the staining mix for bone marrow HSPCs. Shown are volumes of reagents required for the staining of one sample containing 40 million cells. For staining a higher number of samples, multiply the indicated volume by the required number of samples and add half of an extra sample volume to ensure a sufficient volume of the master mix.
Supplementary Figure 1: Bone marrow cell isolation protocol. (A)&#160;Open the peritoneum. White dotted lines indicate the line to cut along. (B)&#160;After peeling the skin off from the hind leg, line the scissors along the spine at the hip joint to cut out the leg without cutting through the femur. (C)&#160;The appearance of the leg separated from the body before the removal of muscle. (D)&#160;The appearance of the leg after removal of muscle. (E)&#160;The procedure for separating the femur at the knee joint, then at the hip joint, taking care not to cut open the femur. White curved arrows show the motion required. White dotted arrows indicate the area to separate gently using scissors to pinch off. (F)&#160;Procedure to open the distal part of the femur (i.e., the part previously attached to the tibia at the knee joint) by securely grabbing the cartilage and distal epiphysis with scissors and flipping it back to expose the bone marrow. (G)&#160;Four protrusions, indicated by black arrows, should be visible at the exposed physis end. (H)&#160;The appearance of a femur with the open-end facing downwards into the prepared 0.5 mL tube placed inside a 1.5 mL tube containing 150 &#181;L of FACS buffer. (I) The appearance of the pelleted bone marrow cells and the now white femur after quick centrifugation at 12,000 x g. <a href="https://www.jove.com/files/ftp_upload/65715/Moraes-Cabe et al_Supplementary Figure1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing Biomedical Research Through Single Cell Analysis at JoVE.com

Author Spotlight: Advancing Biomedical Research Through Single Cell Analysis 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...

high-quality-brain-bone-marrow-nuclei-preparation-for-single-nuclei

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Biology

2.3K Views.  Institut Pasteur, Université Paris Cité. The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay. 

Video: Author Spotlight: Advancing Biomedical Research Through Single Cell Analysis 

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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 ...