Name: optimized nuclei isolation from fresh and frozen solid tumor specimens for multiome sequencing
Uploaded: 2023-10-13T12:20:00.000+00:00
Duration: 3 min 49 s
Description: Watch this Scientific Journal Video about Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments at JoVE.com

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 tumor excision and transport the specimen in quench buffer (DMEM F-12 with ~5% fetal bovine serum) or 1x PBS on ice.</li>
	<li>Mince the tissue using clean forceps and scissors in 3-5 mL of Digest Buffer in a 90 mm Petri dish (Figure 2A).</li>
	<li>Transfer the minced tissue to a 50 mL conical tube and dissociate in ~10 mL of Digest Buffer for 30 min at 37 &#176;C using a water bath with shaking function or a dry agitator.</li>
	<li>Remove from the agitator and quench with ~20 mL of quench medium. Filter the solution through a 100 &#181;m cell strainer into a clean conical tube.</li>
	<li>Collect any remaining solid tissue from the strainer (re-mince remaining tissue pieces if needed), and place in ~10 mL of fresh Digest Buffer for another 30 min at 37 &#176;C with agitation. Repeat until all tumor tissue has been dissociated.</li>
	<li>Pool cell suspension aliquots and filter through a 70 &#181;m followed by a 40 &#181;m cell strainer into a clean conical tube on ice.</li>
	<li>Centrifuge to pellet cells at 500 &#215; g for 5 min at 4 &#176;C and then decant the supernatant.</li>
</ol>
2. Cryopreservation
<ol>
	<li>Rinse the cells by resuspending the pellet in 1 mL of 1x PBS.</li>
	<li>Transfer the cell suspension (~1-10 million cells/tube for optimal freezing) to a 1.5 mL cryovial on ice.</li>
	<li>Centrifuge to pellet cells at 500 &#215; g for 5 min at 4 &#176;C and then decant the supernatant.</li>
	<li>Resuspend the cells in 1 mL of Bambanker solution, transfer to a 1.5 or 2 mL cryovial (Figure 2B), and freeze using a -1 &#176;C/min cooling rate freezing container (containing 100% isopropyl alcohol) at -80 &#176;C, or transfer to liquid nitrogen if longer-term storage of the sample is anticipated.</li>
</ol>
3. Nuclei isolation
<ol>
	<li>Rapidly thaw the cryovial of cell suspension and place it on wet ice.</li>
	<li>Transfer the thawed cell suspension to a 2 mL microcentrifuge tube and top up the vial to 2 mL solution with 1x PBS to rinse the cells.</li>
	<li>Obtain a manual cell count using a hemocytometer to assess both the quantity and quality of the cells (Figure 2C).</li>
	<li>Centrifuge to pellet cells at 500 &#215; g for 5 min at 4 &#176;C and then gently decant the supernatant using progressively smaller pipette tips to leave behind a dry pellet and maintain it on ice.
	<ol>
		<li>Use a swing-bucket rotor for all centrifugation steps. For maximum cell yield, place the tubes in the centrifuge with the hinge facing outwards and then decant with the pipette tip directed towards the opposite side of the tube (Figure 2D). 
		NOTE: For decanting the supernatant, use progressively smaller pipette tips to remove fluid to limit disruption of the pellet while maximizing the volume of fluid decanted. This strategy is particularly helpful later in the protocol following nuclei extraction since the nuclei pellet is generally not visible.</li>
	</ol>
	</li>
	<li>Prepare 1x Cell Lysis Buffer, Cell Lysis Dilution Buffer, and Wash Buffer (Figure 3A) according to the published protocol with the following modifications (see Table 1,&#160;Table of Materials): 
	NOTE: Place the Digitonin on a heating block at 65 &#176;C prior to use to dissolve the precipitate (Figure 3B). This typically takes about 10 min.</li>
	<li>Prepare the 0.1x Cell Lysis Buffer by combining 100 &#181;L of 1x Cell Lysis Buffer and 900 &#181;L of Cell Lysis Dilution Buffer, pipette gently to mix, and place it on ice.</li>
	<li>Resuspend the cell pellet in 100 &#181;L of chilled 0.1x Cell Lysis Buffer and gently pipette to mix 5x.</li>
	<li>Incubate on ice for 3 min (Figure 3C).</li>
	<li>Add 1 mL of chilled Wash Buffer and pipette up and down to gently mix 5x.</li>
	<li>Centrifuge to pellet the nuclei at 500 &#215; g for 5 min at 4 &#176;C, gently decant the supernatant to a dry pellet, and maintain on ice. 
	NOTE: If the starting cell number is low (100,000-200,000 cells), perform the cell lysis and wash steps in a 200 &#181;L PCR tube instead of a 2 mL microcentrifuge tube to decrease the tube surface area and minimize losses. In this case, adjust the Wash Buffer volume down to accommodate the smaller tube volume.</li>
	<li>Repeat Steps 3.9-3.10 2x for a total of three washes.</li>
	<li>Manually count the nuclei using a hemocytometer slide under the microscope. Use trypan blue dye if desired to provide contrast for viewing the nuclei and to help discern nuclei from unlysed cells.</li>
	<li>Prepare the 1x Nuclei Buffer per the published protocol: 20x Nuclei Buffer stock, 1 mM DTT, 1 U/&#181;L RNase Inhibitor in nuclease-free water and keep on ice (see Table of Materials).</li>
	<li>Resuspend the nuclei pellet in 1x Nuclei Buffer based on goal-targeted nuclei recovery. 
	NOTE: If the concentration is high enough, aim for a targeted recovery of 10,000 or slightly higher at this step (3,230-8,060 nuclei/&#181;L) when determining the starting resuspension volume, given the anticipated volume loss of 25 &#181;L and the 20% decrease in concentration with filtering (Step 3.15).</li>
	<li>Gently pass the resuspended nuclei volume through a 40 &#181;m Pipette Tip Cell Strainer and place the nuclei on ice (Figure 3D).</li>
	<li>Recount the nuclei (Figure 4A-C). Dilute the final solution further with Nuclei Buffer if needed.</li>
	<li>Transport the nuclei on ice and immediately proceed with nuclei capture per published protocols.</li>
</ol>

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>

The authors have no conflicts of interest to disclose.

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 isolation from desmoplastic solid tumor tissue specimens. We have found this approach to yield reliable and high-quality data even when cell suspensions are frozen prior to nuclei isolation and capture.
Increasing the albumin concentration throughout the assay is helpful in limiting cell and downstream nuclei clumping, which can be a problem when working with tumor samples. In terms of tissue digestion, digestion kit reagents can also be used with this protocol if desired (see Table of Materials). If used, we would still recommend following the every-30-min digest/quench and buffer exchanges to maximize the yield of viable cells. Following tissue dissociation, red blood cell lysis can be performed per the manufacturer&#39;s protocol. A gradient cell separation protocol can also be performed at the discretion of the researcher (see Table of Materials). We do not perform these protocols routinely, but we have applied such protocols to similar samples without notable differences in results. If significant, small debris is noted after nuclei filtering, Fluorescence-Activated Nuclei Sorting (FANS) can be considered for further purification. A detailed discussion of FANS, including protocol recommendations, is available but beyond the scope of this protocol. Of note, there are protocols in the literature that consider a fixation step prior to cell lysis. However, we prefer to proceed with non-fixed tissue and nuclei as scATAC-seq works best on unfixed tissue9.
For cell lysis, as the RNase inhibitor is quite costly, it is reasonable to scale down the prepared volume of the Cell Lysis Buffer (and the Cell Lysis Dilution Buffer) compared with the volumes provided in the published protocol, if desired, as long as the ratios of reagents stay proportional. When assessing and counting the harvested nuclei, a LIVE/DEAD Viability/Cytotoxicity Kit can be applied at the discretion of the researcher. Trypan blue dye can also be used to provide contrast for viewing the nuclei and to help discern nuclei from unlysed cells if needed. Finally, it is worth noting that we have applied the same cell lysis conditions with adjusted reagents per protocol for single-cell ATAC-seq with excellent results10.
Critical steps in the protocol are precise timing of lysis and gentle but expedient handling of the nuclei. Undermixing will lead to incomplete or inconsistent cell lysis; however, overmixing will shear the chromatin. As noted in other nuclei isolation protocols, nuclei mixing should be accomplished by pipetting rather than vortexing of the sample as the shearing forces can damage the fragile nuclei11. In this regard, it is important that the Cell Lysis Buffer be prepared fresh just prior to use on each occasion. Pipette Tip cell straining (filtering) after the final centrifugation step&#160;is key in our experience to prevent nuclei clumping prior to capture. This filtering can be repeated prior to loading if clumps are still detected on the final count or develop during transport of the specimen prior to loading, keeping in mind the additional loss in volume and concentration with each filtering step added. Of note, there should be no significant time pause between nuclei isolation and capture.
Limitations of this protocol are that tissue dissociation and particularly, the cell lysis incubation timing and conditions may need to be optimized for different solid tumor types12. This protocol has been optimized for desmoplastic solid tumors such as breast carcinoma and pancreatic adenocarcinoma and has also been successfully applied to non-tumor desmoplastic tissues such as parenchymal fibrosis, but other tumor types may require additional adjustments. It is optimized for both fresh cell suspensions as well as frozen cell suspensions with excellent nuclei yield from both of these. We recommend pursuing such optimization sequentially starting with tissue dissociation, and once an optimal single-cell suspension has been achieved, moving on to the optimization of the cell lysis conditions to yield an optimal single-nucleus suspension.
Single-cell ATAC-sequencing and more recently, multiome sequencing present a tremendous breakthrough tool for untangling solid tumor cellular heterogeneity. Cell subtypes can be delineated based on both their chromatin accessibility and gene expression characteristics, and the influence of transcription factor accessibility and expression on tumor behavior can be examined directly. These methodological advances are being broadly applied given their tremendous potential to uncover novel therapeutic targets. As such, the development of optimized protocols to obtain these data is of utmost importance. Here we share this protocol for nuclei isolation in the context of pancreatic cancer tissue-a cancer type for which few therapies exist and all with limited efficacy to date.

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><tbody><tr><td>100, 70, and 40 &amp;mu;m Falcon cell strainers&amp;nbsp;</td><td>&amp;nbsp;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&amp;nbsp;</td><td>Wako, Fisher Scientitic</td><td>NC9582225&amp;nbsp;</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&amp;rsquo;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 &amp;mu;m&amp;nbsp; Pipette Tip Cell Strainer&amp;nbsp;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M1028</td><td></td></tr><tr><td>Miltenyi GentleMACSTM digest kit&amp;nbsp;</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 &quot;Mr. Frosty&quot; Freezing Container</td><td>&amp;nbsp;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&amp;nbsp;</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.

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-D). Representative nuclei images show appropriate size and shape&#160;(Figure 4A-C). The nuclei circled in pale grey show mild stippling of the envelope.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65831/65831fig01.jpg" /> 
Figure 1: Schematic of nuclei isolation workflow. Schematic showing the basic workflow from a whole tumor tissue specimen to a single-nucleus suspension ready for submission for nuclei capture, multiome library preparation, and ultimately scRNA- and ATAC-sequencing. Abbreviations: scRNA = single-cell RNA; ATAC-seq = assay for transposase-accessible chromatin with sequencing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65831/65831fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65831/65831fig02.jpg" /> 
Figure 2: Tissue digestion, cryopreservation, cell assessment, and centrifugation steps. (A)&#160;Equipment setup for mincing tumor tissue specimens; (B)&#160;cell suspension cryopreservation; (C)&#160;microscopic evaluation of cell suspension; (D)&#160;approach to centrifuging for this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65831/65831fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65831/65831fig03.jpg" /> 
Figure 3: Preparation of reagents, nuclei extraction, and filtering. (A) Preparation of nuclei extraction reagents; (B)&#160;dissolution of digitonin at 65 &#176;C prior to use; (C)&#160;incubation of samples for cell lysis on ice; (D) filtering of extracted nuclei. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65831/65831fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65831/65831fig04.jpg" /> 
Figure 4: Representative microscopic images of nuclei harvested from complex tumor tissue specimens. (A-C) Representative images of nuclei harvested with and without Trypan Blue staining. (B,C) The nuclei circled in pale grey show mild stippling of the nuclear envelope. 10x and 40x objectives used. Scale bars = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65831/65831fig04large.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>Solution Name</td>
			<td colspan="3">Components</td>
		</tr>
		<tr>
			<td rowspan="6">Digest Buffer</td>
			<td colspan="3">0.5-1 mg/mL collagenase type IV</td>
		</tr>
		<tr>
			<td colspan="3">100 U/mL DNase I</td>
		</tr>
		<tr>
			<td colspan="3">0.1% Poloxamer 188</td>
		</tr>
		<tr>
			<td colspan="3">20 mM HEPES</td>
		</tr>
		<tr>
			<td colspan="3">1 mM CaCl2</td>
		</tr>
		<tr>
			<td colspan="3">3-5% fetal bovine serum (FBS) in Medium 199</td>
		</tr>
		<tr>
			<td rowspan="9">1X Cell Lysis Buffer</td>
			<td colspan="3">10 mM Tris-HCl (pH 7.4)</td>
		</tr>
		<tr>
			<td colspan="3">10 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">3 mM MgCl2</td>
		</tr>
		<tr>
			<td colspan="3">2% BSA (rather than 1%)</td>
		</tr>
		<tr>
			<td colspan="3">0.10% Tween-20&#160;</td>
		</tr>
		<tr>
			<td colspan="3">0.1% Nonident P40 Substitute</td>
		</tr>
		<tr>
			<td colspan="3">0.01% Digitonin</td>
		</tr>
		<tr>
			<td colspan="3">1 mM DTT</td>
		</tr>
		<tr>
			<td colspan="3">1 U/&#181;L RNAse inhibitor in Nuclease-free water</td>
		</tr>
		<tr>
			<td rowspan="6">Cell Lysis Dilution Buffer</td>
			<td colspan="3">10 mM Tris-HCl (pH 7.4)</td>
		</tr>
		<tr>
			<td colspan="3">10 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">&#160;3 mM MgCl2</td>
		</tr>
		<tr>
			<td colspan="3">2% BSA</td>
		</tr>
		<tr>
			<td colspan="3">1 mM DTT</td>
		</tr>
		<tr>
			<td colspan="3">1 U/&#181;L RNAse inhibitor in Nuclease-free water</td>
		</tr>
		<tr>
			<td rowspan="7">Wash Buffer</td>
			<td colspan="3">10 mM Tris-HCl (pH 7.4)</td>
		</tr>
		<tr>
			<td colspan="3">10 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">3 mM MgCl2</td>
		</tr>
		<tr>
			<td colspan="3">2% BSA</td>
		</tr>
		<tr>
			<td colspan="3">0.10% Tween-20</td>
		</tr>
		<tr>
			<td colspan="3">1 mM DTT</td>
		</tr>
		<tr>
			<td colspan="3">1 U/&#181;L RNAse inhibitor in Nuclease-free water</td>
		</tr>
	</tbody>
</table>
Table 1: Solutions used in this protocol.

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

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

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.

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) ...

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Nanyang Technological University

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JoVE Journal

Cancer Research

947 Views.  Stanford University. Naarmate onze kennis van tumorbiologie groeit en onze interesse om de fijne kneepjes te begrijpen van hoe cellen in de tumor interageren en de resultaten van de patiënt beïnvloeden, is ook het belang van het analyseren van heterogene cellen in de micro-omgeving van de tumor toegenomen. Multioomsequencing, die het mogelijk maakt om op een gepaarde celmanier RNA van één cel en aanvalssequencing van dezelfde cel te verwerven, biedt een belangrijke vooruitgang in de richting van dit doel. De ...

Video: Auteur in de schijnwerpers: Verbetering van de isolatie van kernen voor multioomsequencing in uitdagende tumormicro-omgevingen

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

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 ...

Biology

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

High-Quality Sample Preparation for Multiome 10X Genomics Analysis

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.

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.

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

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

Source: Narayanan, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61542/nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus">Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.</a> J. Vis. Exp. (2020)
This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
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Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

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