Aug 27, 2020
HTAPP_Dissociation of human neuroblastoma tumors to a single-cell suspension for single-cell RNA-seq using Papain
- Anand Patel1,
- Asa Karlstrom1,
- Brittney Gordon1,
- Elizabeth Stewart1,
- Michael Dyer1
- 1St. Jude Children's Research Hospital
- NCIHTAN
Protocol Citation: Anand Patel, Asa Karlstrom, Brittney Gordon, Elizabeth Stewart, Michael Dyer 2020. HTAPP_Dissociation of human neuroblastoma tumors to a single-cell suspension for single-cell RNA-seq using Papain. protocols.io https://dx.doi.org/10.17504/protocols.io.98ah9se
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 06, 2019
Last Modified: August 27, 2020
Protocol Integer ID: 30690
Keywords: Neuroblastoma, single-cell RNA-sequencing, dissociation of fresh pediatric neuroblastoma sample, fresh pediatric neuroblastoma sample, htapp-dissociation of human neuroblastoma tumor, human neuroblastoma tumor, neuroblastoma resection, processing other tumor, human tumor atlas pilot project, cell suspensions compatible with droplet, cell rna, cell suspension, rna
Abstract
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 (Slyper et al).
For the Human Tumor Atlas Pilot Project (HTAPP), this protocol has been successfully applied to neuroblastoma resections collected from abdominal and paraspinal sites.
Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found in Slyper et al.
Guidelines
Store papain kit at 4° C.
Materials
MATERIALS
PDS Kit, Papain VialWorthington Biochemical CorporationCatalog #LK003178
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
Aspen Surgical™ Bard-Parker™ Protected Disposable Scalpel No. 10Fisher ScientificCatalog #02-688-78
A1000 PipettorMIDSCICatalog #A-1000
Pipet-aid pipette controllerDrummond ScientificCatalog #4-000-110
Falcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
AvantGuard 1250 microliter filter tipsMIDSCICatalog #AV1250-H
Falcon 5mL sterile serological pipetsFisher ScientificCatalog #13-675-22
Falcon 10mL sterile serological pipetsFisher ScientificCatalog #13-675-20
Sterile 40 micron nylon strainerFisher ScientificCatalog #08-771-1
Trypan blue 0.4% solutionFisher ScientificCatalog #15-250-061
1.5 mL microcentrifuge tubesFisher ScientificCatalog #05-408-129
Thermo Scientific Sorvall Legend XTR CentrifugeFisher ScientificCatalog #75-217-420
Bright-Line HemacytometerFisher ScientificCatalog #02-671-5
A100 pipetterCatalog #A-100
AvantGuard 100 microliter filter tipsMIDSCICatalog #AV1000
Falcon 60 mm tissue-culture treated dishesFisher ScientificCatalog #08-772B
Troubleshooting
Safety warnings
Follow general lab safety and institutional guidelines for working with human samples and sharps.
Before start
- Warm water bath to 37° C.
- Allow EBSS (vial #1), albumin-ovomucoid inhibitor mixture (vial #4), papain (vial #2) and DNase I (vial #3) to warm to room temperature for 15 min.
- If you are starting with a new kit, add 32 ml of Earle's balanced salt solution (EBSS; PDS kit vial #1) to albumin-ovomucoid inhibitor (vial #4).
Reagent Preparation
Prepare reagents (1 set for each sample):
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
00:15:00
15m
Add 500 µL of Earle's Balanced Salt Solution (vial #1) to DNase I (vial #3). Mix using micropipette.
Room temperature
Add 250 µL from DNase I solution (vial #3) to papain solution (vial #2).
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).
Room temperature Process tissue quickly to avoid viability loss (mincing should take less than 5 min)
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.
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.
37 °C Water bath
00:15:00
Triturate tissue by pipetting up and down 10-15 times using a 10 mL sterile serologic pipette.
Room temperature
Continue dissociation by incubating tube in the water bath for another 15 min.
37 °C Water bath
00:15:00
Repeat trituration by pipetting up and down 10-15 times using a 10 mL sterile serologic pipette.
Room temperature
Note: The cell suspension should look cloudy with chunks of debris.
Filter cell suspension through a 40 µm cell strainer into a 50 mL Falcon conical tube.
Room temperature
Note: Debris will be captured on the cell strainer, while the filtrate should appear cloudy.
Rinse filter with 5 mL Earle's Balanced Salt Solution (vial #1).
Centrifuge cell suspension at 500 g for 5 min at room temperature.
500 x g, 00:05:00
Room temperature
Cleanup
During centrifugation, prepare the two 15 mL Falcon conical tubes containing:
Resuspension buffer:
2.7 mL Earle's Balanced Salt Solution (vial #1)
300 µL Albumin-ovomucoid inhibitor solution (vial #4)
150 µL DNase I solution (vial #3)
Room temperature
Density gradient:
5 mL Albumin-ovomucoid inhibitor solution (vial #4)
Room temperature
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.
Room temperature
Re-suspend pellet with 3 mL resuspension buffer (from step #10.1), and gently mix using a 5 mL serologic pipette.
Room temperature
Carefully layer resuspended cells over density gradient (from step #10.2).
Room temperature
Centrifuge at 100 g for 6 min at room temperature.
100 x g, 00:06:00
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.
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.
Room temperature
Mix 10 µL of single-cell suspension with 10 µL of Trypan blue solution, and load 10 µL onto hemacytometer.
Room temperature
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.
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.
Keep diluted cell suspension on ice, and proceed immediately to 10x Genomics single-cell RNA-sequencing workflow.
On ice Proceed immediately to 10x Genomics RNA-seq workflow