TITLE: HTAPP_Dissociation of human neuroblastoma tumors to a single-cell suspension for single-cell RNA-seq using Papain
AUTHORS: Anand Patel, Asa Karlstrom, Brittney Gordon, Elizabeth Stewart, Michael Dyer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was used for the dissociation of fresh pediatric neuroblastoma samples from patient tumors or orthotopic patient derived xenografts. Using this method, we were able to prepare highly viable (>90%) single-cell suspensions compatible with droplet-based single-cell RNA-Seq technology (</span><a href="https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41591-020-0844-1&data=01%7C01%7CAnand.Patel2%40stjude.org%7Cdd690a388cde4ae4b04808d84aac46f3%7C22340fa892264871b677d3b3e377af72%7C0&sdata=taMGWERtsEjyZbKOGJ%2B%2Fxf%2Bws%2FNGheB3tKaZhJGS9Hs%3D&reserved=0" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Slyper et al</span></a><span>).</span></div><div class = "text-block">For the Human Tumor Atlas Pilot Project (HTAPP), this protocol has been successfully applied to neuroblastoma resections collected from abdominal and paraspinal sites.</div><div class = "text-block"><span>Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found in </span><a href="https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41591-020-0844-1&data=01%7C01%7CAnand.Patel2%40stjude.org%7Cdd690a388cde4ae4b04808d84aac46f3%7C22340fa892264871b677d3b3e377af72%7C0&sdata=taMGWERtsEjyZbKOGJ%2B%2Fxf%2Bws%2FNGheB3tKaZhJGS9Hs%3D&reserved=0" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Slyper et al</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Reagent Preparation]
Prepare reagents (1 set for each sample):
?. [Reagent Preparation]
Add 5 mL of Earle's Balanced Salt Solution (vial #1) to papain (vial #2). Allow papain to dissolve for 15 min at 37° C.
37 °C
?. [Reagent Preparation]
Add 500 µL of Earle's Balanced Salt Solution (vial #1) to DNase I (vial #3). Mix using micropipette.
0 Room temperature
?. [Reagent Preparation]
Add 250 µL from DNase I solution (vial #3) to papain solution (vial #2).
0 Room temperature
?. [Tissue dissociation]
Obtain tumor tissue. Use 100-500 mg of tissue per papain vial. Use a disposable scalpel to mince the sample into small pieces (approximately 1 mm fragments).Note: We typically receive tissue on ice from the repository. Tissue is processed immediately upon receipt. Once dissociation has started, we do not recommend putting tissue back onto ice (temperature fluctuations lead to tissue degradation). Note: We typically mince tissue on a 60 mm sterile tissue-culture treated plate.
[Process tissue quickly to avoid viability loss (mincing should take less than 5 min)]
?. [Tissue dissociation]
Move minced tissue into a sterile 15 mL Falcon conical tube, and transfer papain-DNase mixture from vial #2 to the conical tube. Incubate conical tube in water bath at 37° C for 15 min.
[Water bath]
?. [Tissue dissociation]
Triturate tissue by pipetting up and down 10-15 times using a 10 mL sterile serologic pipette.
0 Room temperature
?. [Tissue dissociation]
Continue dissociation by incubating tube in the water bath for another 15 min.
[Water bath]
?. [Tissue dissociation]
Repeat trituration by pipetting up and down 10-15 times using a 10 mL sterile serologic pipette.Note: The cell suspension should look cloudy with chunks of debris.
0 Room temperature
?. [Tissue dissociation]
Filter cell suspension through a 40 µm cell strainer into a 50 mL Falcon conical tube.Note: Debris will be captured on the cell strainer, while the filtrate should appear cloudy.
0 Room temperature
?. [Tissue dissociation]
Rinse filter with 5 mL Earle's Balanced Salt Solution (vial #1).
?. [Tissue dissociation]
Centrifuge cell suspension at 500 g for 5 min at room temperature.
Centrifuge: 500 34, 5 min
0 Room temperature
?. [Cleanup]
During centrifugation, prepare the two 15 mL Falcon conical tubes containing:
?. [Cleanup]
After centrifugation (step #9), remove supernatant carefully without disturbing the pellet. Tip: We typically use suction to aspirate the first 9 mL of supernatant, followed by a micropipette to remove the last 0.5-1 mL of supernatant (to avoid suctioning the pellet). If the pellet is very small or difficult to see, we recommend transferring the top 9.5 mL of supernatant to a tube labeled 'supernatant 1' as a backup.
0 Room temperature
?. [Cleanup]
Re-suspend pellet with 3 mL resuspension buffer (from step #10.1), and gently mix using a 5 mL serologic pipette.
0 Room temperature
?. [Cleanup]
Resuspension buffer:
[Earle's Balanced Salt Solution (vial #1)]
[Albumin-ovomucoid inhibitor solution (vial #4)]
[DNase I solution (vial #3)]
0 Room temperature
?. [Cleanup]
Density gradient:
[Albumin-ovomucoid inhibitor solution (vial #4)]
0 Room temperature
?. [Cleanup]
Carefully layer resuspended cells over density gradient (from step #10.2).
0 Room temperature
?. [Cleanup]
Centrifuge at 100 g for 6 min at room temperature.
Centrifuge: 100 34, 6 min
0 Room temperature
?. [Cell counting and Quality Control]
Carefully remove supernatant without disturbing the pellet.Tip: We typically use suction to aspirate the first 7 mL of supernatant, followed by a micropipette to remove the last 0.5-1 mL of supernatant (to avoid suctioning the pellet). If the pellet is very small or difficult to see, we recommend transferring the top 7.5 mL of supernatant to a tube labeled 'supernatant 2' as a backup.
?. [Cell counting and Quality Control]
Mix 10 µL of single-cell suspension with 10 µL of Trypan blue solution, and load 10 µL onto hemacytometer.
0 Room temperature
?. [Cell counting and Quality Control]
Count and report the number of viable single cells.Tip: We recommend proceeding only with samples that have >80% viability. Low viability samples will have higher cell clumping, and ambient RNA will complicate downstream single-cell RNA-sequencing analysis.
0 Room temperature
?. [Cell dilution and 10x Genomics single-cell RNA-seq loading]
If necessary, dilute cells prior to loading onto 10x Genomics 3' single cell RNA-sequencing workflow. Tip: We aim to load 10,000 cells per channel. We recommend trying to dilute cells to a concentration 1000 cells/µL.
?. [Cell dilution and 10x Genomics single-cell RNA-seq loading]
Keep diluted cell suspension on ice, and proceed immediately to 10x Genomics single-cell RNA-sequencing workflow.
[Proceed immediately to 10x Genomics RNA-seq workflow]
?. [Cell counting and Quality Control]
Re-suspend pellet in 10 mL Earle's Balanced Salt Solution and filter through a 40 µm cell strainer into a sterile 50 mL Falcon conical tube.
0 Room temperature