This study was supported by the Adelson Medical Research Foundation (AMRF), the Melanoma Research Alliance (MRA), and U54CA224068. Figure 1, Figure 4, and Figure 5 were created with BioRender.com.

This study complied with all institutional guidelines regarding human tissue sampling. Informed consent was received from patients, and identifiable sample data was anonymized. Samples are collected in the operation room, placed in a solution of RPMI, saline, or PBS, and confirmed cancerous via the pathology department before use in research. All steps, except when indicated, should be carried out at 4 &#176;C or on ice. Work inside a biosafety hood when processing human tissue. See the Table of Materials for details on all materials, reagents, and instruments used in this protocol.
1. Obtaining the sample
<ol>
	<li>Obtain a solid tumor sample in a tube of media and place it on ice.
	<ol>
		<li>Instruct the operating room staff to transport the sample in a tube containing enough RPMI, saline, or PBS to completely submerge the tissue to avoid sample dehydration. 
		NOTE: Ensuring the sample is not desiccated is important, as this will decrease viability.</li>
	</ol>
	</li>
	<li>Cool down all centrifuges to 4 &#730;C and bring the thermal mixer to 37 &#730;C.</li>
	<li>Take out prepared human or mouse tumor dissociation enzymes (Table of Materials), thaw on the 37 &#730;C thermal mixer, and move to ice once thawed.</li>
	<li>Retrieve a 20 mL aliquot of RPMI.</li>
	<li>Record relevant sample information (e.g., Patient ID, de-identifier, sample type).</li>
	<li>Transfer the sample to a 60 mm tissue culture dish (placed on ice) by inverting the tube several times and pouring the contents into the dish to ensure all sample material is transferred. If the sample was not delivered in media upon arrival, add fresh RPMI media to the dish.
	<ol>
		<li>If the sample is fragmented, spin the tube containing the sample at 450 &#215; g, 4 &#730;C for 5 min, discard the supernatant, resuspend the sample in 420 &#181;L of RPMI, and move to section 2.</li>
	</ol>
	</li>
</ol>
2. Mechanical and enzymatic digestion
<ol>
	<li>Pipette 420 &#181;L of media from the 60 mm sample dish to a 1.5 mL microcentrifuge tube. Attempt to include any sample fragments suspended in the media.</li>
	<li>Transfer the sample tissue from the 60 mm dish to the 1.5 mL tube containing media using tweezers. Cut up the sample with clean scissors inside the tube so the pieces are maximum 2-3 mm in diameter.</li>
	<li>Add digestion enzymes, prepared according to manufacturer specifications (Table of Materials), to the sample in the following volumes: 42 &#181;L of Enzyme H (nonspecific proteases) for human samples, Enzyme D (nonspecific proteases) for murine samples, 21 &#181;L of enzyme R (nonspecific proteases), and 5 &#181;L of enzyme A (nuclease).</li>
	<li>Add another 420 &#181;L of media from the dish or up to the 1 mL mark on the tube. Do not exceed 420 &#181;L of media.</li>
	<li>Vortex the tube for 5 s and place it in a thermal mixer positioned vertically on its side (Figure 1B). Incubate at 37 &#730;C, 450 rpm, for 15 min. 
	NOTE: During incubation, take out all 10x Genomics materials needed for GEM generation, as these require 30 min to equilibrate to room temperature</li>
</ol>
3. Sample filtering
<ol>
	<li>Place a 50 &#181;m cell filter on top of a new 15 mL conical tube and prime the filter by passing 1 mL of fresh RPMI media through it into the tube.</li>
	<li>After digestion, transfer the sample to the 15 mL tube using a 1,000 &#181;L low-retention wide pipette tip. Wash the 1.5 mL tube with 1 mL of fresh media and transfer the media to the filter to ensure all sample material is passed through the filter.</li>
	<li>Obtain a 1 mL plastic syringe and take out just the plunger (discard the remainder of the syringe). Using the back of the syringe plunger, grind and push the sample on the filter in a twisting motion.</li>
	<li>Wash the filter with 5 mL of media and any remaining media from the dish that contained the sample (section 1) to collect any cells that might have detached from the tissue.</li>
	<li>Place a 30 &#181;m cell filter on top of a new 15 mL conical tube, prime the filter with 1 mL of media, and transfer the contents of the tube containing the dissociated sample through the filter. 
	NOTE: This step will remove large debris that might clog or cause a wetting failure when running the sample using the 10x Genomics microfluidic chips.</li>
</ol>
4. Red blood cell lysis
<ol>
	<li>Centrifuge the sample at 450 &#215; g, 4 &#730;C for 5 min. Aspirate the media, being careful not to disturb the pellet.</li>
	<li>Resuspend in ACK Lysis buffer to remove red blood cells (RBCs) and record the volume used. The volume will depend on pellet size and the extent of RBCs in the pellet. 
	NOTE: Resuspended ACK-sample solution should be colorless when enough ACK Lysis buffer has been added, as this will clear the red blood cells from the sample.</li>
	<li>Centrifuge the sample tube at 450 &#215; g, 4 &#730;C for 5 min, and aspirate the supernatant.</li>
	<li>Repeat steps 4.2-4.3 as needed until the sample pellet has no visible red color after centrifugation (Figure 2). Record any extra volumes of ACK Lysis buffer used in this step.</li>
	<li>Resuspend the sample pellet in fresh media in preparation for counting.</li>
</ol>
5. Cell counting
<ol>
	<li>Place 10 &#181;L of trypan blue in a 1.5 mL tube. Gently mix the dissociated cells, take 10 &#181;L of sample, and mix (1:1) with the trypan blue. Load 10 &#181;L on each side of a hemocytometer and count the cells.</li>
	<li>Record cell concentration, total live cell count, viability percentage, and final media volume after count.</li>
	<li>Ensure cell count is between 700 and 1,200 cells/&#181;L (7 &#215; 105- 1.2 &#215; 106/mL) for an optimal 10x Genomics run.
	<ol>
		<li>If the sample is too concentrated, dilute it with media and recount it.</li>
		<li>If the sample is too diluted, centrifuge the sample at 450 &#215; g, 4 &#730;C for 5 min, and resuspend in a lesser volume of media.</li>
		<li>If cell viability is below 80%, perform a live-dead procedure to remove dead cells.</li>
		<li>If there are under one million cells, see section 6.</li>
		<li>If there are over one million cells and viability&#160;&#62; 10%, see section 6.</li>
		<li>If there are over one million cells and viability &#60; 10%, see section 7.</li>
		<li>If cell viability is above 80%, keep the processed cells on ice and proceed immediately to GEM generation according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>
6. Dead cell removal - Kit 1
NOTE: Work inside a biosafety hood when using the dead cell removal reagents, as these can easily get contaminated.
<ol>
	<li>Centrifuge the sample at 450 &#215; g, 4 &#730;C for 5 min.</li>
	<li>Prepare isolation media in a 15 mL conical tube and keep it at room temperature: 9.8 mL of PBS, 10 &#181;L of CaCl2, and 200 &#181;L of heat-inactivated FCS (added in a Biosafety hood).</li>
	<li>After centrifugation, aspirate the media from the pellet and resuspend in 1 mL of the isolation media.</li>
	<li>Centrifuge the tube again at 450 &#215; g, 4 &#730;C for 5 min. Aspirate the supernatant and resuspend the pellet in 100 &#181;L of isolation media.</li>
	<li>Transfer 100 &#181;L of sample in isolation media into one well of a 0.2 mL PCR strip-tube.</li>
	<li>In a biosafety hood, add 10 &#181;L of Annexin V Cocktail and 10 &#181;L of Biotin Selection Cocktail to the well containing 100 &#181;L of the sample in isolation media. Pipette mix the solution and allow it to sit at room temperature for 4 min.</li>
	<li>After 4 min, vortex the dextran-coated magnetic particles for 30 s. Add 20 &#181;L of the beads and 60 &#181;L of isolation media to the sample (a total volume of 200 &#181;L). If viability is lower than 40%, scale the amount of beads up to 40 &#181;L while adjusting the volume of isolation media (up to 40 &#181;L), keeping a total volume of 200 &#181;L. Pipette mix and allow the solution to sit at room temperature for 3 min.</li>
	<li>Place the strip tube onto a 10x Genomics Magnetic Separator (on the high setting) for 5 min at room temperature.</li>
	<li>Transfer the liquid from the strip tube without disturbing the beads into a new 1.5 mL lo-bind microcentrifuge tube. Do not remove the strip-tube from the magnet.</li>
	<li>Centrifuge the 1.5 mL tube containing the sample at 450 &#215; g, 4 &#730;C for 5 min. Aspirate the supernatant and resuspend the pellet in an appropriate volume of RPMI media based on the pellet size and count cells, as described in section 5. Repeat the dead cell removal procedure if viability remains below 80% (Figure 3 and Figure 4). 
	NOTE: Do not resuspend cells in isolation media for downstream GEM generation, as the calcium chloride will interfere with the encapsulation process and the reverse transcription (RT) step.</li>
	<li>Keep processed cells on ice if cell viability is above 80% and proceed immediately to GEM generation according to the manufacturer&#39;s instructions.</li>
</ol>
7. Dead cell removal - Kit 2
NOTE: Work inside a biosafety hood when using the kit reagents.
<ol>
	<li>Place the microbeads and the 20x Binding Buffer stock solution inside a biosafety hood.</li>
	<li>Centrifuge the sample suspension at 450 &#215; g, 4 &#730;C for 5 min.</li>
	<li>Prepare 1x buffer solution under sterile conditions using 1 mL of the 20x Binding Buffer stock solution pipetted into 19 mL of DNAse-, RNAse-free distilled water.</li>
	<li>Once the centrifugation is completed, aspirate the supernatant, add the microbeads, gently mix the sample, and incubate at room temperature for 15 min.
	<ol>
		<li>When the cell count is 107 or less, add 100 &#181;L of the microbeads.</li>
		<li>For counts higher than 107 total cells, adjust the microbeads for a final concentration of 100 &#181;L per 107 live cells.</li>
	</ol>
	</li>
	<li>During incubation, obtain one MACS positive selection column, MACS multistand, and the corresponding magnetic separator. Ensure the separator is leveled on the multistand and no other metal or magnetic object is near the stand, as the magnetic force is powerful and could affect dead cell removal efficiency. Place the column snugly into a separator holder with wings facing out.</li>
	<li>After sample incubation, if the total resuspension volume is less than 500 &#181;L, add 1x Binding Buffer until the total volume is 500 &#181;L.</li>
	<li>Place a 15 mL conical tube below the column and prime the column with 3 mL of 1x Binding Buffer.</li>
	<li>Replace the conical tube with a new 15 mL conical for cell sample collection and add the sample to the column, allowing it to flow completely through the column.</li>
	<li>Wash the column 4 x 3 mL of 1x Binding Buffer, only adding one wash at a time after the previous wash entirely flowed through the column.</li>
	<li>After the fourth wash, centrifuge the conical tube containing the isolated cells for 5 min at 450 &#215; g, 4 &#730;C. Discard the column and the remaining 1x Binding Buffer.</li>
	<li>Resuspend the sample solution in the appropriate amount of RPMI media based on pellet size and count the isolated cells using the method described in section 5 (Figure 3 and Figure 5).
	<ol>
		<li>If viability remains below 80%, and the total number of cells is 106 or less, repeat section 6.</li>
		<li>If cell viability is above 80%, keep processed cells on ice and proceed immediately to GEM generation according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>

Tumor Dissociation Protocol for Single-Cell RNA Sequencing Analysis

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H., Bang, D. <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+technologies+and+bioinformatics+pipelines.">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Exp Mol Med. 50 (8), 1-14 (2018).</li><li>Erfanian, N., 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=Deep+learning+applications+in+single-cell+genomics+and+transcriptomics+data+analysis.">Deep learning applications in single-cell genomics and transcriptomics data analysis.</a> Biomed Pharmacother. 165, 115077 (2023).</li><li>Jovic, D., 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+technologies+and+applications:+A+brief+overview.">Single-cell RNA sequencing technologies and applications: A brief overview.</a> Clin Transl Med. 12 (3), e694 (2022).</li><li>Burja, B., 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=An+optimized+tissue+dissociation+protocol+for+single-cell+RNA+sequencing+analysis+of+fresh+and+cultured+human+skin+biopsies.">An optimized tissue dissociation protocol for single-cell RNA sequencing analysis of fresh and cultured human skin biopsies.</a> Front Cell Dev Biol. 10, 872688 (2022).</li><li>Denisenko, 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=Systematic+assessment+of+tissue+dissociation+and+storage+biases+in+single-cell+and+single-nucleus+RNA-seq+workflows.">Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows.</a> Genome Biol. 21 (1), 130 (2020).</li><li>Sade-Feldman, 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=Defining+T+cell+states+associated+with+response+to+checkpoint+immunotherapy+in+melanoma.">Defining T cell states associated with response to checkpoint immunotherapy in melanoma.</a> Cell. 176 (1-2), 404 (2019).</li><li>Pelka, K., 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=Spatially+organized+multicellular+immune+hubs+in+human+colorectal+cancer.">Spatially organized multicellular immune hubs in human colorectal cancer.</a> Cell. 184 (18), 4734-4752.e20 (2021).</li><li>Bill, R., 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=CXCL9:SPP1+macrophage+polarity+identifies+a+network+of+cellular+programs+that+control+human+cancers.">CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.</a> Science. 381 (6657), 515-524 (2023).</li><li>Al'Khafaji, A. 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=High-throughput+RNA+isoform+sequencing+using+programmed+cDNA+concatenation.">High-throughput RNA isoform sequencing using programmed cDNA concatenation.</a> Nat Biotechnol. 42 (4), 582-586 (2024).</li><li>Xing, Y., 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=The+effect+of+cell+isolation+methods+on+the+human+transcriptome+profiling+and+microbial+transcripts+of+peripheral+blood.">The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.</a> Mol Biol Rep. 48 (4), 3059-3068 (2021).</li><li>Ma, F., Hernandez, G. E., Romay, M., Iruela-Arispe, M. 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M.S-F. received funding from Bristol Myers-Squibb, Istari Oncology, and was a consultant for Galvazine Therapeutics.

This protocol describes human or murine tumor samples dissociation into a single-cell suspension for scRNA sequencing using the 10x Genomics microfluidic system. In processing tissues that often come from rare cancers or are part of ongoing clinical trials or long experiments for murine tumors, the sample must be optimally and carefully preserved between all steps. All steps should be carried out rapidly on ice or at 4 &#730;C to keep the native cellular RNA profiles and prevent degradation, as working at room temperatur...

Although many advancements in cancer research have led to the development and FDA approval of agents targeting inhibitory receptors expressed on immune and tumor cells, known as checkpoint blockade inhibitors, therapy resistance and identifying mechanisms that drive patient response remain challenging1. The complex challenge of characterizing tumor heterogeneity on its molecular diversity and the intricate interplay between tumor cells and the immune microenvironment necessitates new approaches to dissect this complex ecosystem at the single-cell resolution. Understanding the molecular intricacies within tumors and their microenvironments is pivotal for advancing therapeutic strategies and deciphering the complex biology underlying cancer progression2. Single-cell RNA sequencing (scRNA-seq) has become increasingly popular because it provides high-resolution analysis of individual cells within complex tumor samples3,4.
Tumor heterogeneity, encompassing genetic, epigenetic, and phenotypic diversities, poses a challenge in uncovering the intricacies of cancer biology5. The conventional methods of bulk RNA sequencing tend to obscure the unique expression profiles and phenotypes of heterogeneous cell populations present within tumors, as these methods average signals across entire cell populations4. In contrast, scRNA-seq allows the dissection of individual cell types, revealing diverse gene expression, repression patterns, and cellular states otherwise overlooked4,6. The premise of scRNA-seq lies in its capacity to decode individual cells&#39; genetic and molecular signatures, holding immense promise in discovering new cancer therapeutics7.
By deciphering the gene expression profiles of tumor cells and the surrounding stromal and immune cells, researchers can identify novel therapeutic targets and develop precision genetic medicine strategies tailored to individual patients6. Furthermore, dissecting the immune repertoire within the tumor microenvironment provides crucial insights into the interplay between tumor cells and immune effectors, paving the way for immunotherapeutic interventions3. Despite advances in using scRNAseq on clinical samples, several challenges during the processing steps can harm the cell&#39;s RNA integrity, viability, and quality of the data generated. Moreover, processing tissue with low cell viability can increase ambient RNA levels in the droplets generated during the encapsulation of individual cells, resulting in cross-contamination and incorrect annotation of cell types, while long dissociation protocols performed at 37 &#176;C can lead to the upregulation of a stress response gene signature in multiple cell types8,9,10.
The stepwise procedure (Figure 1A) outlined in this protocol commences with rapid mechanical and enzymatic dissociation of tumors, followed by filtration and the removal of dead cells, depending on the viability of each tumor sample. At present, this procedure has been verified in melanoma11, colorectal12, head and neck squamous cell carcinoma13, pancreatic and lung (unpublished data) tumor samples. These techniques ensure the generation of high-quality viable cell suspensions crucial for downstream analyses14. Notably, removing red blood cells, devoid of synthesized RNA, becomes imperative to purify the sample15. Subsequent evaluation of cell counts and the elimination of dead cells serve as a prerequisite for successful 10x Genomics Gel bead-in Emulsion (GEM) generation, barcoding, and increase the recovery of transcripts and sequenced cells without jeopardizing the exclusion of sensitive populations (e.g., neutrophils and epithelial cells)16. These steps are fundamental in unlocking the potential of single-cell resolution analysis within tumor samples9,10, uncovering novel gene expression profiles and immune signatures necessary for driving innovative therapeutic interventions, and identifying new tumor vulnerabilities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L low-retention wide pipette tips</td>
			<td>Rainin</td>
			<td>30389218</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389212</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Magnetic Separator</td>
			<td>10x Genomics</td>
			<td>120250</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL aspirating pipette</td>
			<td>Corning</td>
			<td>357558</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389226</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389225</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389240</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389239</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>60 x 15 mm Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A10492-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>3090659</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>551660</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Solution</td>
			<td>Sigma Aldrich</td>
			<td>2115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Ranger&#160;</td>
			<td>10x Genomics</td>
			<td>N/A</td>
			<td>https://www.10xgenomics.com/support/software/cell-ranger/latest</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC20 cell counter</td>
			<td>Bio-Rad</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics 30&#956;m sterile disposable filters</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics 50&#956;m sterile disposable filters</td>
			<td>Sysmex</td>
			<td>04-004-2327</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell removal Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td>Store at -20 &#176;C; kit 2</td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip</td>
			<td>VWR</td>
			<td>82027-386</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Microcentrifuge tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySepTM Dead Cell Removal 
			(Annexin V) Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17899</td>
			<td>Store at 4 &#176;C; Kit 1</td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5425R</td>
			<td>Eppendorf</td>
			<td>5406000640</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5910R</td>
			<td>Eppendorf</td>
			<td>2231000657</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Easypet 3 Electronic Pipette controller</td>
			<td>Eppendorf</td>
			<td>EP4430000018</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Thermomixer F1.5 Model 5384</td>
			<td>Eppendorf</td>
			<td>EP5384000012</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>Store at 4 &#176;C, use in a Biological hood</td>
		</tr>
		<tr>
			<td>German Stainless Scissors</td>
			<td>Fine Science Tools</td>
			<td>14568-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Dmi1 Microscope</td>
			<td>Leica Microsystems</td>
			<td>454793</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadro MACS Magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-051</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 (1x)</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>TempAssure 0.2 mL PCR 8-Tube strips</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit, Human</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>Store at -20 &#176;C, prepare enzymes according to kit instructions: 
			Reconstitute lyophilized Enzyme H vial in 3 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.</td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit, Mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-730</td>
			<td>Store at -20 &#176;C, prepare enzymes according to kit instructions: 
			Reconstitute lyophilized Enzyme D vial in 3 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>Store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

dissociation of human and mouse tumor tissue samples for single-cell rna sequencing

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq).
The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.

Author Spotlight: Exploring Strategies for Successful Immune Response Against Tumors

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

The scope of our research is to study the mechanisms of response or resistance in standard of care immunotherapies. Our lab is trying to understand what drives a successful immune response against tumors, and if we can identify these predictors of therapy to find targets that can overcome resistance. The biggest advancement in recent years has been the development and integration of single-cell RNA sequencing techniques.They enable us in real time to systematically study molecular features in individual cells in an unbiased manner, which is helpful in the dissection of these complex and evolving tumor ecosystems. While many preclinical models are being used to study the response mechanisms to immunotherapy, we still don't know the immunological determinants that are important in human immunity. Processing and analyzing patient samples in real time is crucial to our ability to investigate all cellular compartments within a given tumor.Using unbiased genomic and transcriptomic techniques, our lab discovered that defects in the antigen presentation machinery are associated with immunotherapy resistance in melanoma. More recently, using single-cell approaches, we discovered unique T cell states and myeloid cell polarities associated with patient outcomes in melanoma and head neck cancer, respectively. The biggest advantage of our protocol lies in its simplicity.It is a universal protocol adjusted for the dissociation of small and large tumor specimens from multiple types of cancers in a short period of time, ending in the generation of highly-viable, single-cell suspensions.

A melanoma biopsy suspended in RPMI was obtained and immediately placed on ice. The sample was transferred into a 1.5 mL microcentrifuge tube containing 420 &#181;L of RPMI and digestion enzymes, minced into small pieces using scissors, and subjected to subsequent enzymatic digestion for 15 min in a 37 &#730;C vertically positioned thermal mixer (Figure 1). Following digestion, the sample was filtered through a 50 &#181;m sterile disposable filter, and the filter was washed with fresh RPMI t...

This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into ...

dissociation-human-mouse-tumor-tissue-samples-for-single-cell-rna

Research

JoVE Journal

Cancer Research

Rapid Dissociation of Tumor Tissue to Gain Enhanced Single-Cell Tumor Insights

To begin tumor digestion, pour the sample into a 60-millimeter tissue culture dish placed on ice. Pipette 420 microliters of media from the dish to a 1.5-milliliter microcentrifuge tube. Using tweezers, transfer the sample tissue to the tube containing media.Cut up the sample with clean scissors to pieces that are two to three millimeters in diameter. Add appropriate volumes of digestion enzymes and media to the sample. Vortex the tube for five seconds, and incubate it in a thermal mixer positioned vertically on its side.Place a 50-micrometer cell filter on top of a new 15-milliliter conical tube. And prime the filter with one milliliter of fresh RPMI media. Using a 1, 000-microliter lower tension wide pipette tip, load the sample to the filter and add the media.Then, with the back of a syringe plunger, grind and push the sample on the filter in a twisting motion. Wash the filter with five milliliters of media and any remaining media from the dish that contain the sample.

Red Blood Cell Lysis in Digested Tumor Samples for Single-Cell RNA Sequencing

To begin, obtain the filtered and digested tumor sample. Centrifuge the sample at 450 G for five minutes at four degrees Celsius. Aspirate the media without disturbing the pellet and resuspend the pellet in ACK lysis buffer to remove red blood cells and record the volume used.Repeat the centrifugation and the lysis steps until the sample pellet has no visible red color after centrifugation. Then resuspend the sample pellet in fresh media. Mix 10 microliters each of trypan and blue and the dissociated cells in a 1.5 milliliter tube.Load 10 microliters on each side of a hemocytometer. to count the cells. Record the cell concentration.Total live cell count viability percentage, and final media volume after count.

Dead Cell Removal from Dissociated Tumor for Single-Cell Sequencing

To begin, obtain the digested tumor tissue sample and lice the red blood cells. Centrifuge the sample at 450G for five minutes at four degrees Celsius. After removing the media, resuspend the pellet in one milliliter of the isolation media.After repeating the centrifugation, resuspend the pellet in 100 microliters of isolation media. Transfer 100 microliters of sample into a well of a 0.2 milliliter PCR strip tube. In a biosafety hood, add 10 microliters each of annexin V cocktail and biotin selection cocktail to the sample.Pipette mix the solution and incubate at room temperature for four minutes. Next, vortex the dextran-coated magnetic particles for 30 seconds. Mix 20 microliters of the beads and 60 microliters of isolation media with the sample.After three minutes, place the strip tube onto a 10x Genomics magnetic separator on the high setting for five minutes at room temperature. Then transfer the liquid from the strip tube without disturbing the beads into a new 1.5 milliliter low bind micro centrifuge tube. Centrifuge the tube as demonstrated earlier, and resuspend the pellet in an appropriate volume of our PMI media based on the pellet size.Finally, count the cells in a hemocytometer. Following dead cell removal, the live cell count was 4.9 times 10 to the power of 5 per milliliter, with viability above 90%

2.4K Views.  Massachusetts General Hospital.  This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

Tumor Dissociation Protocol for Single-Cell RNA Sequencing Analysis

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