We would like to acknowledge the Stanford Functional Genomics Facility (SFGF), particularly Dhananjay Wagh and John Coller, and 10x Genomics for their assistance with optimizing our experiments. We would also like to thank Drs. George Poultsides, Monica Dua, Brendan Visser, and Byrne Lee for their assistance in acquiring patient specimens. We would like to acknowledge Art and Elaine Taylor, the Rantz Foundation, and Warren and Judy Kaplan for their generous support of our research efforts. Funding sources include NIH grants 1F32CA23931201A1 (D.S.F.), 1R01GM116892 (M.T.L.), 1R01GM136659 (M.T.L), Goldman Sachs Foundation (J.A.N., D.S.F., M.T.L.), the Damon Runyon Cancer Research Foundation (D.D., M.T.L.), the Gunn/Olivier Fund, the California Institute for Regenerative Medicine, Stinehart/Reed Foundation, and the Hagey Laboratory for Pediatric Regenerative Medicine. Sequencing was obtained using machines purchased with NIH funds (S10OD025212, S10OD018220, and 1S10OD01058001A1).

Human pancreatic cancer (pancreatic ductal adenocarcinoma) samples were acquired according to an IRB-approved protocol in our laboratory. Informed consent was obtained from patients for tissue collection. Tissue was transported from the operating room to the laboratory and then processed as follows.
1. Tissue dissociation (digestion)
<ol>
	<li>Prepare Digest Buffer (see Table of Materials).</li>
	<li>Obtain the tissue of interest as soon as possible after tu...

Isolation of Nuclei for Multiome Sequencing in Solid Tumor Specimen

<ol>
	<li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+and+spatial+transcriptomics+reveal+cancer-associated+fibroblasts+in+glioblastoma+with+protumoral+effects.">Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects.</a> J Clin Invest. 133 (5), e147087 (2023).</li><li>Sahai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+advancing+our+understanding+of+cancer-associated+fibroblasts.">A framework for advancing our understanding of cancer-associated fibroblasts.</a> Nat Rev Cancer. 20 (3), 174-186 (2020).</li><li>Foster, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiomic+analysis+reveals+conservation+of+cancer-associated+fibroblast+phenotypes+across+species+and+tissue+of+origin.">Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin.</a> Cancer Cell. 40 (11), 1392-1406 (2022).</li><li>Hasan, S., Buechler, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+in+a+name?+An+emerging+framework+for+cancer-associated+fibroblasts,+myofibroblasts,+and+fibroblasts.">What's in a name? An emerging framework for cancer-associated fibroblasts, myofibroblasts, and fibroblasts.</a> Cancer Cell. 40 (11), 1273-1275 (2022).</li><li>. Nuclei isolation from complex tissues for single cell multiome ATAC + gene expression sequencing Available from: <a target="_blank" href="https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-complex-tissues-for-single-cell-multiome-atac-plus-gene-expression-sequencing">https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-complex-tissues-for-single-cell-multiome-atac-plus-gene-expression-sequencing</a> (2022)</li><li>. Nuclei isolation from embryonic mouse brain tissue for single cell multiome ATAC + gene expression sequencing Available from: <a target="_blank" href="https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-embryonic-mouse-brain-tissue-for-single-cell-multiome-atac-plus-gene-expression-sequencing">https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-embryonic-mouse-brain-tissue-for-single-cell-multiome-atac-plus-gene-expression-sequencing</a> (2022)</li><li>Januszyk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+diabetic+and+non-diabetic+foot+ulcers+using+single-cell+RNA-sequencing.">Characterization of diabetic and non-diabetic foot ulcers using single-cell RNA-sequencing.</a> Micromachines (Basel). 11 (9), 815 (2020).</li><li>Foster, D. S., Jones, R. E., Ransom, R. C., Longaker, M. T., Norton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+relationship+of+wound+healing+and+tumor+stroma.">The evolving relationship of wound healing and tumor stroma.</a> JCI Insight. 3 (18), e99911 (2018).</li><li>Nott, A., Schlachetzki, J. C. M., Fixsen, B. R., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclei+isolation+of+multiple+brain+cell+types+for+omics+interrogation.">Nuclei isolation of multiple brain cell types for omics interrogation.</a> Nat Protoc. 16 (3), 1629-1646 (2021).</li><li>Foster, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+spatial+multiomics+reveals+fibroblast+fate+during+tissue+repair.">Integrated spatial multiomics reveals fibroblast fate during tissue repair.</a> Proc Natl Acad Sci U S A. 118 (41), e2110025118 (2021).</li><li>Leiz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclei+isolation+from+adult+mouse+kidney+for+single-nucleus+RNA-sequencing.">Nuclei isolation from adult mouse kidney for single-nucleus RNA-sequencing.</a> J Vis Exp. (175), (2021).</li><li>Narayanan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclei+isolation+from+fresh+frozen+brain+tumors+for+single-nucleus+RNA-seq+and+ATAC-seq.">Nuclei isolation from fresh frozen brain tumors for single-nucleus RNA-seq and ATAC-seq.</a> J Vis Exp. (162), e61542 (2020).</li></ol>

Untangling the heterogeneous cell populations present in the tumor microenvironment is an active area of focus in cancer biology. Similarly, complex tissues exist in benign pathologies such as wound healing and fibrosis. Multiome sequencing has emerged as a powerful tool permitting the acquisition of same-cell paired scRNA- and ATAC-seq data. This protocol describes the isolation of nuclei, which demands optimization in the setting of processing fresh, fragile, small tumor specimens. Here we provide a protocol for nuclei...

As our knowledge of tumor biology grows, the importance of analyzing heterogeneous cells across the tumor microenvironment has also increased1,2. The ability to acquire single-cell RNA and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data from the same cell in a paired-cell fashion (multiome sequencing) provides a significant advance towards this end3,4. These experiments are expensive and time-consuming, however, and the quality and impact of the data acquired are highly dependent on the quality of the experimental conditions and materials. Standardized protocols for nuclei isolation have been published5,6. Fresh and heterogeneous tissues require protocol optimization since freshly isolated cells from solid tumor specimens are more fragile than those isolated from cell lines.
Another consideration is that for solid tumors, surgical specimens are often not available from the operating room until late in the day. As such, it is generally not feasible to proceed directly from sample acquisition to nuclei capture without a cryopreservation step. In our experience, freezing a single-cell suspension yields the highest-quality nuclei (rather than flash-frozen whole tissue or other modalities of preservation). This is particularly true for enzymatic tissue types with high RNase content such as the pancreas.
Tissue digestion conditions also need to be designed to maximize cell yield without sacrificing quality7. In the context of solid tumor types with dense desmoplastic matrices8, the extracellular matrix must be gently broken down for cell release. Additionally, because the cell types isolated are heterogeneous, conditions should be adjusted to support the total cell population. Human pancreatic cancer (pancreatic ductal adenocarcinoma) samples are used in the described protocol. Pancreatic cancer represents a highly desmoplastic tumor type, which portends relatively sticky tissue and cells. Moreover, as pancreatic tumor specimens available for research also tend to be relatively small, efforts are made to maximize the quantity of cells captured.
Isolation of nuclei requires the most optimization in terms of cell lysis conditions and timing, as well as reagent types and ratios. Handling the nuclei over the course of isolation also requires great care. In this article, we describe our experience optimizing nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor tissue (Figure 1). We provide recommendations for tissue digestion, cryopreservation of single-cell suspensions (if desired), and nuclear isolation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100, 70, and 40 &#956;m Falcon cell strainers&#160;</td>
			<td>&#160;ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Genomics Nuclei Buffer (20x)</td>
			<td>10x Genomics</td>
			<td>2000153/2000207</td>
			<td></td>
		</tr>
		<tr>
			<td>Bambanker&#160;</td>
			<td>Wako, Fisher Scientitic</td>
			<td>NC9582225&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (Collagenase Type IV)</td>
			<td>ThermoFisher</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Thermo Fisher</td>
			<td>BN2006</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington</td>
			<td>LS006330</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium F-12</td>
			<td>Thermo Fisher</td>
			<td>11320082</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmi 40 &#956;m&#160; Pipette Tip Cell Strainer&#160;</td>
			<td>Sigma Aldrich</td>
			<td>BAH136800040</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque-1119 Gradient Cell Separation solution</td>
			<td>Sigma Aldrich</td>
			<td>11191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma Aldrich</td>
			<td>M2520</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Miltenyi GentleMACSTM digest kit&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>59222C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Cryo &#34;Mr. Frosty&#34; Freezing Container</td>
			<td>&#160;ThermoFisher</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonident P40 Substitute</td>
			<td>Sigma Aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Poloxamer 188</td>
			<td>Sigma</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase inhibitor&#160;</td>
			<td>Sigma Aldrich</td>
			<td>3335399001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma Aldrich</td>
			<td>T2194</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Thermo Fisher</td>
			<td>85113</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized nuclei isolation from fresh and frozen solid tumor specimens for multiome sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

As our knowledge of tumor biology grows and interest to understand the intricacies of how cells interact within the tumor and influence patient outcomes, the importance of analyzing heterogeneous cells across the tumor microenvironment has also increased. Multiome sequencing, which permits the acquisition of single cell RNA and attack sequencing from the same cell in a paired cell fashion provides a significant advance towards this end. However, the quality of the data obtained from these experiments is highly dependent on the quality of the experimental conditions and inputted biological material.This protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform. This protocol is reliable using either fresh or frozen single cell suspensions. We provide recommendations for tissue dissociation conditions, cryopreservation of single cell suspensions, and assessment of isolated nuclei.In this protocol, we used human pancreatic ductal adenocarcinoma specimens, which are a primary tissue type of focus in our laboratory. Pancreas cancer represents a highly desmoplastic tumor type, which portends relatively sticky tissue as well as cells. Moreover, as pancreatic tumor specimens available for research also tend to be relatively small, efforts are made to maximize the quantity of the cells captured.

To isolate high-quality nuclei from patient solid tumor specimens for multiome sequencing (Figure 1), the tumor tissue was dissociated and a single-cell suspension was cryopreserved (Figure 2A-D). The cell suspension was then thawed at the time of planned multiome capture. Nuclei capture was conducted with optimized lysis buffer reagents and timing to maximize both quality and yield (Figure 3A<stron...

Watch this Scientific Journal Video about Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments at JoVE.com

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

optimized-nuclei-isolation-from-fresh-frozen-solid-tumor-specimens

Research

JoVE Journal

Cancer Research

Nuclei Isolation from Human Pancreatic Tumor Cells for Multiome Sequencing

Begin by transferring previously thawed pancreatic tumor cell suspension to a two-milliliter microcentrifuge tube and add PBS to reach a final volume of two milliliters. Then, use a hemocytometer to evaluate the quantity and quality of the cells. Once the cell pellet is obtained through centrifugation, using progressively smaller pipette tips, carefully decant the supernatant to minimize pellet distribution while maximizing the fluid decanted.Prepare 1X cell lysis buffer, then add 100 microliters of it to 900 microliters of previously prepared cell lysis dilution buffer to obtain 0.1X cell lysis buffer. Transfer 100 microliters of chilled 0.1X cell lysis buffer to the cell pellet and gently pipette five times to ensure complete resuspension. After incubation on ice for three minutes, add one milliliter of chilled wash buffer to the resuspended pellet and gently pipette it five times.Following centrifugation, discard the supernatant as demonstrated, and repeat this washing process two more times. Load trypan blue-stained suspension into a hemocytometer to count the number of nuclei. Resuspend the nuclei pellet in 1X nuclei buffer based on goal-targeted nuclei recovery.Carefully pass the resuspended nuclei through a 40-micrometer pipette tip cell strainer, and then maintain it on ice. Now recount the nuclei. The nuclei obtained using this method exhibited appropriate size and shape.Occasional mild stippling of the nuclear envelope can be observed and should be monitored at the final nuclear evaluation via hemocytometer.

744 Views.  Stanford University.  The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei. 

Isolation of Nuclei for Multiome Sequencing in Solid Tumor Specimen

Summary