TITLE: HTAPP_Dissociation of human metastatic breast cancer core needle biopsy to a single-cell suspension for single-cell RNA-seq
AUTHORS: Michal Slyper, Julia Waldman, Jingyi Wu, Abhay Kanodia, Sébastien Vigneau, Asaf Rotem, Bruce Johnson, Orit Rozenblatt-Rosen, Aviv Regev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used for the dissociation of human metastatic breast cancer core needle biopsies to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology (</span><a href="https://www.nature.com/articles/s41591-020-0844-1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Slyper et al</span></a><span>).</span></div><div class = "text-block">For the Human Tumor Atlas Pilot Project (HTAPP) it has been successfully applied to biopsies collected from breast, liver and lymph node.</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://www.nature.com/articles/s41591-020-0844-1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Slyper et al</span></a><span style = ":;font-style:italic;">.</span></div></div>

[STEPS]
?. [Sample Description]
Report sample processing information.
[Wet Ice]
Sample ID:Date:Anatomical Site of the Biopsy:Number of Biopsy Cores:Core(s) Priority Number:Media Used for Transportation:Person Processing:
?. [Sample Description]
Transfer the sample to a Petri dish with cold PBS (or RPMI without phenol red) kept on ice in order to better visualize its composition. Take a picture of each core alongside a ruler, and describe its appearance (e.g., is it fragmented or necrotic?).
[Wet Ice]
Describe Sample Appearance:Core(s) Dimensions (mm):Insert Picture(s) of Sample:
?. [Tissue Dissociation]
Transfer the sample to a 2 mL screw cap tube (e.g., cryotube) or Eppendorf tube containing 1 mL cold dissociation mix per core. Report time at which dissociation starts.Tip: The suggested amount of 1 mL per core works well on 18 gauge (~0.8 mm diameter), 10-20 mm long cores, but may be reduced or increased for smaller or larger cores, respectively. If the volume of dissociation mix is larger than 1.5 mL, dissociation may be performed in a 5 mL (instead of 2 mL) Eppendorf tube.
[Wet Ice]
Volume of Dissociation Mix Used (mL):Dissociation Start Time:
?. [Tissue Dissociation]
Mince the sample with spring scissors to Tip: If spring scissors are not available, scalpels can be used instead. In this case, the biopsy should be minced in a Petri dish within
[Proceed quickly at room temperature]
?. [Tissue Dissociation]
Incubate for 10 minutes at 37˚C, with rotation at approximately 14 rpm.Tip: If using an Eppendorf tube, wrap the tube's 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 1 mL tip.Tip: Do not put the tube on ice as repeated, drastic changes in temperature may be detrimental to cell viability. Pipette down pushing against the wall of the tube for optimal dissociation. If fragments are too large for pipetting, continue mincing with spring scissors before pipetting.
[Proceed quickly at room temperature]
?. [Tissue Dissociation]
Incubate for another 10 minutes at 37˚C, with rotation at approximately 14 rpm.Tip: If using an Eppendorf tube, wrap the tube's lid with Parafilm to prevent any leakage. Use lab tape to secure tubes on the rotator.
[Enzymatic Dissociation, Part 2]
[Hybridization Oven, 14 rpm]
?. [Tissue Dissociation]
Continue dissociation by pipetting up and down 20-30 times using a pipette with 1 mL tip. No or only very small fragments should be visible by eye and the solution should appear cloudy.Tip: Pipette down pushing against the wall of the tube for optimal dissociation. If fragments are too large for pipetting, continue mincing with spring scissors before pipetting.
[Proceed quickly at room temperature]
?. [Tissue Dissociation]
Centrifuge at 300 g for 4 minutes in 4˚C pre-cooled centrifuge. This long spin helps to get rid of fat and debris more efficiently than subsequent short spins.
[Centrifugation]
[Centrifuge]
?. [Red Blood Cell Removal]
Report the cell pellet color (e.g., red, pink, white) in the first row of the table at Step 16.
[Wet Ice]
?. [Red Blood Cell Removal]
Carefully transfer the supernatant to the 15 mL “Supernatant 1” tube kept on ice, making sure to remove any fat and to not 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.
[Wet Ice]
?. [Red Blood Cell Removal]
Resuspend the cell pellet in 200-500 µL cold ACK Lysing Buffer to lyse red blood cells.Tip: The volume of ACK 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.
[Wet Ice]
?. [Red Blood Cell Removal]
Incubate for 1 minute on ice.
[ACK Red Blood Cell Lysis]
[Wet Ice]
?. [Red Blood Cell Removal]
Mix in a volume of PBS equal to twice the volume of 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 1.7 mL Eppendorf tube (500 µL - 1 mL) and proceed quickly to the next step.
[Wet Ice]
?. [Red Blood Cell 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.
[Centrifugation]
[Centrifuge]
?. [Red Blood Cell Removal]
If the cell pellet is pink or red, revealing a significant portion of red blood cells, repeat steps 11-15. 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 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
?. [Cell Clump Dissociation]
Carefully transfer the supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
[Wet Ice]
?. [Cell Clump Dissociation]
Resuspend the cell pellet in 100 µL TrypLE per core.
[Wet Ice]
?. [Cell Clump Dissociation]
Hold tube in hand for 1 min while constantly pipetting up and down using a pipette with 200 µL tip.Tip: Pipette down pushing against the wall or bottom of the tube for optimal dissociation.
[TrypLE Treatment]
?. [Cell Clump Dissociation]
Inactivate TrypLE by mixing with 200 µL of cold RPMI 1640 with 10% FBS per 100 µL TrypLE.
[Wet Ice]
?. [Cell Clump Dissociation]
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.
[Centrifugation]
[Centrifuge]
?. [Cell Clump Dissociation]
Carefully transfer the supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
[Wet Ice]
?. [Cell Clump Dissociation]
Resuspend the cell pellet in 50 µL cold PBS with 0.4% BSA.
[Wet Ice]
?. [Quality Control]
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on hemocytometer.
?. [Quality Control]
Count and report the number of viable single cells, dead single cells, cell doublets or clumps, and whether debris are present, then calculate additional Quality Control metrics below. Take picture if possible.  ABC1Initial Quality ControlQuality Control after Optional Debris and Cell Clumps Removal (Step 26)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 26)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 26):
?. [[Optional] Debris and Cell Clumps Removal]
If the quantity of debris or cell clumps is too high to load on 10x Genomics Single-Cell RNA-seq system and the number of cells is at least double from 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 the cell pellet in 500 µL cold PBS with 0.4% BSA.
[Wet 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 to the tube. Flow cytometry tubes with 35 µm cell strainer snap cap can also be used but typically result in lower cell recovery.
[Wet 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.
[Wet Ice]
?. [[Optional] Debris and Cell Clumps Removal]
Transfer to Eppendorf tube.
[Wet 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.
[Centrifugation]
[Centrifuge]
?. [[Optional] Debris and Cell Clumps Removal]
Carefully transfer the supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
[Centrifuge]
?. [[Optional] Debris and Cell Clumps Removal]
Resuspend the cell pellet in 50 µL cold PBS with 0.4% BSA.
[Centrifuge]
?. [[Optional] Debris and Cell Clumps Removal]
Repeat steps 24-25.
[Quality Control]
?. [Loading on 10x Genomics Single-Cell RNA-seq System]
If necessary, adjust the concentration before proceeding to loading on 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%.
[Wet Ice]
?. [Loading on 10x Genomics Single-Cell RNA-seq System]
Report the information listed below about loading on 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: