TITLE: HTAPP_Dissociation of primary neuroblastoma resection to a single-cell suspension for single-cell RNA-seq (using papain, density gradient, and optionally ACK)
AUTHORS: Sara Napolitano, Jingyi Wu, Michal Slyper, Sébastien Vigneau, Asaf Rotem, Bruce Johnson, Orit Rozenblatt-Rosen, Aviv Regev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was used for the dissociation of human neuroblastoma resections to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology for the Human Tumor Atlas Pilot Project. For processing human neuroblastoma tumors, however, we now recommend using the papain-based protocol established by </span><a href="http://dx.doi.org/10.17504/protocols.io.98ah9se" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Patel et al</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Tissue Description]
Report sample processing information.
Sample ID:Date:Time Received:Media Used for Transportation:Person Processing:
?. [Tissue Description]
Transfer sample to a Petri dish with cold PBS kept on ice to better visualize its composition. Take a picture of the resection alongside a ruler and annotate its different regions. Tumors are typically stiff and light-colored. Necrotic regions tend to be soft and crumbly.
on ice
Describe Sample Appearance:Insert Picture(s) of Sample:
?. [Tissue Dissociation]
Transfer the tumor piece or fragments to a 5 mL Eppendorf tube containing 3 mL cold Papain and DNAse solution. Report time at which dissociation starts.Tip: The suggested amount of 3 mL works well on 25-200 mm3 resections but may be reduced or increased for smaller or larger resections, respectively.
on ice
Volume of Dissociation Mix Used (mL):
Dissociation Start Time:
?. [Tissue Dissociation]
Mince the biopsy with spring scissors into Tip: If spring scissors are not available, scalpels can be used instead. In this case, the tumor piece should be minced in a Petri dish with 1-2 mL of the dissociation mix before transferring the resulting tissue fragment suspension to a tube with the remaining dissociation mix.
[Proceed quickly]
?. [Tissue Dissociation]
Incubate for 12:30 minutes at 37˚C, with rotation at approximately 14 rpm. Tip: If using an Eppendorf tube, wrap the lid with Parafilm to prevent any leakage. Use lab tape to secure tubes on the rotator.
[Enzymatic Dissociation, Part 1]
[Hybridization Oven, 14 rpm]
?. [Tissue Dissociation]
Continue dissociation by pipetting up and down 20-30 times using a pipette with a 1 mL tip.Tip: Do not put the tube on ice as repeated drastic temperature changes may be detrimental to cell viability. If fragments are too large for pipetting, continue mincing with spring scissors or enlarge the 1 mL tip's opening by cutting its extremity with scissors or a scalpel.
[Proceed quickly]
?. [Tissue Dissociation]
Incubate for another 12:30 minutes at 37˚C, with rotation at approximately 14 rpm.
[Enzymatic Dissociation, Part 2]
[Hybridization Oven, 14 rpm]
?. [Tissue Dissociation]
Continue dissociation by pipetting up and down until the tissue disaggregates, which may take up to 5 minutes. Start by using a pipette with a regular 1 mL tip, then continue using a 1 mL tip bent 2-3 mm from its extremity to increase tissue shearing. No or only very small fragments should be visible by eye, and the solution should appear cloudy.Tip: Do not put the tube on ice as repeated, drastic changes in temperature may be detrimental to cell viability. If fragments are too large for pipetting, continue mincing with spring scissors or enlarge the opening of the 1 mL tip by cutting its extremity with scissors or a scalpel. If the tissue does not disaggregate, additional enzymatic dissociation may be added by 5 minutes increments.
20 Room temperature
?. [Tissue Dissociation]
Centrifuge at 300 g for 5 minutes at room temperature. This long spin helps to get rid of fat more efficiently than subsequent short spins.
Centrifuge: 300 34, 5 min, 4 32
?. [Cleanup with Discontinuous Density Gradient]
During the centrifugation, prepare the DNase Albumin Ovomucoid Inhibitor Solution as follows:
[EBSS (Vial 1)]
[Albumin Ovomucoid Inhibitor Solution (Vial 4)]
[DNase Solution (Vial 3)]
20 Room temperature
?. [Cleanup with Discontinuous Density Gradient]
Carefully transfer supernatant to the 15 mL “Supernatant 1” tube kept on ice, making sure to remove any fat that may be floating on top and not to disturb the cell pellet.Tip: If any fat is visible, aspirate and discard it before transferring the remaining of the supernatant using a different pipette tip.
4 Room temperature
?. [Cleanup with Discontinuous Density Gradient]
Resuspend cell pellet in 3 mL of the DNase Albumin Ovomucoid Inhibitor Solution prepared in Step 14.
20 Room temperature
?. [Cleanup with Discontinuous Density Gradient]
Prepare discontinuous density gradient by adding 5 mL of Albumin Ovomucoid Inhibitor Solution (Vial 4) into a 15 mL conical tube and carefully layering the cell suspension on top.
20 Room temperature
?. [Cleanup with Discontinuous Density Gradient]
Centrifuge at 70 g for 6 minutes at 4˚ C.
Centrifuge: 70 34, 6 min
?. [Cleanup with Discontinuous Density Gradient]
Report the cell pellet color (e.g., red, pink, white) in the first row of the table at Step 25. If the cell pellet is red or pink, indicating the presence of red blood cells, proceed to the optional red blood cell removal with ACK Lysing Solution (Step 20). If the cell pellet is white, continue to Quality Control (Step 26).
on ice
?. [[Optional] Red Blood Cell Removal with ACK Lysing Solution]
Carefully transfer supernatant to the 15 mL “Supernatant 1” tube kept on ice, making sure to not disturb the cell pellet.
on ice
?. [[Optional] Red Blood Cell Removal with ACK Lysing Solution]
Resuspend the cell pellet in 300-500 µL cold ACK Lysing Buffer to lyse red blood cells. If cells are in a 15 mL tube, transfer them to a 1.5 mL Eppendorf tube kept on ice.Tip: The ACK volume should be adjusted to the size and color of the cell pellet and may be increased up to 1 mL if the cell pellet is large or extremely bloody.
on ice
?. [[Optional] Red Blood Cell Removal with ACK Lysing Solution]
Incubate for 1 minute on ice.
[ACK Red Blood Cell Lysis]
on ice
?. [[Optional] Red Blood Cell Removal with ACK Lysing Solution]
Mix in a PBS volume equal to twice the volume ACK Lysing Buffer and proceed quickly to the next step.Tip: If more than 500 µL ACK Lysing Buffer was used, mix in the largest volume of PBS that can safely fit in a 1.5 mL Eppendorf tube (500 µL - 1 mL) and proceed quickly to the next step.
on ice
?. [[Optional] Red Blood Cell Removal with ACK Lysing Solution]
Centrifuge for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to (but not exceeding) 11,000 g. Proceed quickly to the next step.Tip:  Do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death.
Centrifuge: 11000 34, 8 s, 4 10
?. [[Optional] Red Blood Cell Removal with ACK Lysing Solution]
If the cell pellet is pink or red, revealing a significant portion of red blood cells, repeat Steps 20-24. For each round of ACK Lysing Buffer treatment, report the ACK Lysing Buffer volume, treatment duration, and cell pellet color in the table below.Tip: Avoid exceeding three rounds of 1 min ACK Lysing Buffer treatment, as this may result in an important loss of cell viability.  ABCD1RepeatACK Lysing Buffer Volume (µL)ACK Lysing Buffer Treatment Duration (min)Cell Pellet Color20NANA345
[if pink or red pellet]
ABCD1RepeatACK Lysing Buffer Volume (µL)ACK Lysing Buffer Treatment Duration (min)Cell Pellet Color20NANA345
?. [Quality Control]
Carefully transfer supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
on ice
?. [Quality Control]
Resuspend the cell pellet in 50 µL cold PBS with 0.4% BSA.
on ice
?. [Quality Control]
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on a hemocytometer.
?. [Quality Control]
Count and report the number of viable single cells, dead single cells, cell doublets or clumps, and whether debris are present. Take a picture if possible.  ABC1Initial Quality ControlQuality Control after Optional Debris and Cell Clumps Removal (Step 30)2Number of Viable Single Cells Counted3Number of Dead Single Cells Counted4Number of Cell Clumps or Doublets Counted5Concentration of Viable Single Cells (cells/µL)6Concentration of Dead Single Cells (cells/µL)7Concentration of Cell Clumps or Doublets (doublets/µL)8Volume of Single Cell Suspension (µL)9Total Number of Viable Single Cells10Proportion of Single Cells that are Viable (%)11Proportion of Cell Clumps or Doublets (%)12Description of debris (if any)
ABC1Initial Quality ControlQuality Control after Optional Debris and Cell Clumps Removal (Step 30)2Number of Viable Single Cells Counted3Number of Dead Single Cells Counted4Number of Cell Clumps or Doublets Counted5Concentration of Viable Single Cells (cells/µL)6Concentration of Dead Single Cells (cells/µL)7Concentration of Cell Clumps or Doublets (doublets/µL)8Volume of Single Cell Suspension (µL)9Total Number of Viable Single Cells10Proportion of Single Cells that are Viable (%)11Proportion of Cell Clumps or Doublets (%)12Description of debris (if any)
Insert Picture for Initial Quality Control:Insert Picture for Final Quality Control (if additional cleanup was performed as described in Step 30):
?. [[Optional] Debris and Cell Clumps Removal]
If the quantity of debris or cell clumps is too high to load on the 10x Genomics Single-Cell RNA-seq system and the number of cells is at least double what is required to load, strain the sample as described below.Tip: Samples should typically have less than 5% cell clumps. If enough cells (e.g., at least four times the number of cells to be loaded), strain only half of the sample and keep the other half as a backup.
?. [[Optional] Debris and Cell Clumps Removal]
Resuspend in 500 µL cold PBS with 0.4% BSA.
on ice
?. [[Optional] Debris and Cell Clumps Removal]
Filter through a 30 µm MACS SmartStrainer into a 15 mL tube.Tip: To maximize cell recovery, pipette residual liquid from underneath the strainer and transfer it to the tube. Flow cytometry tubes with a 35 µm cell strainer snap cap can also be used but typically result in lower cell recovery.
on ice
?. [[Optional] Debris and Cell Clumps Removal]
Wash filter with an additional 500 µL of cold PBS with 0.4% BSA to recover as many cells as possible.
on ice
?. [[Optional] Debris and Cell Clumps Removal]
Transfer to Eppendorf tube.
on ice
?. [[Optional] Debris and Cell Clumps Removal]
Centrifuge for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to (but not exceeding) 11,000 g. Proceed quickly to the next step.Tip:  Do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death.
Centrifuge: 11000 34, 8 s, 4 10
?. [[Optional] Debris and Cell Clumps Removal]
Carefully transfer supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
on ice
?. [[Optional] Debris and Cell Clumps Removal]
Resuspend in 50 µL cold PBS with 0.4% BSA.
on ice
?. [[Optional] Debris and Cell Clumps Removal]
Repeat steps 28-29.
[Quality Control]
?. [Loading on 10x Genomics Single-Cell RNA-seq System]
If necessary, adjust the concentration before proceeding to load on the 10x Genomics Single-Cell RNA-seq system, following 10x Genomics recommendations.Tip: 8,000-10,000 live cells are typically loaded per channel. Optimal cell recovery is achieved for concentrations between 800 and 1,200 cells/µL but deviations from that range are acceptable (see 10x Technical Note on this topic). Furthermore, it is recommended that viability be higher than 60% and the proportion of cell clumps lower than 5%.
on ice
?. [Loading on 10x Genomics Single-Cell RNA-seq System]
Report the information listed below about loading on the 10x Genomics Single-Cell RNA-seq system, including the number and concentration of cells per channel.
Time of Loading:   Person Loading:   Single-Cell RNA-seq Kit Used:   Concentration of Viable Cells Loaded (cells/µL):   Number of Cells Loaded per Channel:   Number of Channels Loaded:
?. [Tissue Description]
Transfer the resection to a Petri dish with cold PBS kept on ice and dissect the tumor from non-tumor tissue using a scalpel. Describe the decisions taken to dissect the sample and document them with an annotated picture.
on ice
Describe Tumor Dissection:Dimensions of Dissected Tumor (mm):  Insert Annotated Picture(s) Documenting Tumor Dissection:
?. [Tissue Description]
If required, divide the tumor using a scalpel and allocate pieces to different assays following each assay's requirements. Recommended dimensions to obtain enough cells for the 10x Genomics Single-Cell RNA-seq system are 3x3x3 mm or larger. Describe the decisions taken to allocate the sample, including each piece's dimensions, and document the allocation process with an annotated picture.
on ice
Describe Sample Allocation:
Dimensions of Tissue Piece Allocated (mm):  Insert Annotated Picture(s) Documenting Sample Allocation:
?. [Tissue Dissociation]
If the tumor piece's dimensions are greater than 3x3x3 mm, cut into smaller fragments using safety scalpels.
on ice
?. [Tissue Dissociation]
Filter through a 70 µm cell strainer set on a 15 mL conical tube kept on ice to get rid of fragments. Wash strainer with 5 mL cold PBS.Tip: If the mixture appears to have a lot of undigested tissue pieces that may clog the 70 µm strainer, use a 100 µm strainer instead.
on ice