TITLE: HTAPP_Dissociation of human primary lung cancer resection to a single-cell suspension for single-cell RNA-seq
AUTHORS: Michal Slyper, Avinash Waghray, Julia Waldman, Isaac Wakiro, Mei-Ju Su, Sébastien Vigneau, Asaf Rotem, Bruce Johnson, Orit Rozenblatt-Rosen, Aviv Regev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for the dissociation of human primary lung cancer resections to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology.</div><div class = "text-block"><span>For the Human Tumor Atlas Pilot Project (HTAPP) it has been successfully applied to non-small cell adenocarcinoma from which it was able to capture a diversity of cell types, including malignant (epithelial) and non malignant cells such as  mesenchymal, endothelial, myeloid, and lymphoid cells. In some instances, this protocol can be combined with a </span><a href="http://dx.doi.org/10.17504/protocols.io.bjxjkpkn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocol for CD45-cell depletion</span></a><span>, which is used for fast depletion of immune cells for enrichment of malignant and stromal cells. </span></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 = ":;">Slyper et al</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Sample Description and Allocation]
Report sample processing information.
[Wet Ice]
Sample ID:
Date:Media Used for Transportation:
Person Processing:
?. [Sample Description and Allocation]
Transfer sample to a Petri dish with cold PBS (or HBSS without phenol red) 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, often with dark spots in lung cancer. Necrotic regions tend to be soft and crumbly.
[Wet Ice]
Describe Sample Appearance:
Insert Annotated Picture(s) of Sample:
?. [Sample Description and Allocation]
Transfer the resection to a Petri dish with cold HBSS 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.
[Wet Ice]
Describe Tumor Dissection:Dimensions of Dissected Tumor (mm):
Insert Annotated Picture(s) Documenting Tumor Dissection:
?. [Sample Description and Allocation]
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 10x Genomics Single-Cell RNA-seq system are 3x3x3 mm or larger. Describe the decisions taken to allocate the sample, including the dimensions of each piece, and document the allocation process with an annotated picture.
[Wet Ice]
Describe Sample Allocation:Dimensions of Tissue Piece Allocated (mm):
Insert Annotated Picture(s) Documenting Sample Allocation:
?. [Tissue Dissociation]
If the dimensions of the tumor piece are greater than 3x3x3 mm, cut into smaller fragments using safety scalpels.
[Wet Ice]
?. [Tissue Dissociation]
Transfer the tumor piece or fragments to a 5 mL Eppendorf tube containing 3 mL cold dissociation mix. 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.
[Wet Ice]
Volume of Dissociation Mix Used (mL):Dissociation Start Time:
?. [Tissue Dissociation]
Mince the tissue with spring scissors into Tip: If spring scissors are not available, scalpels can be used instead. In this case, the sample should be minced in a Petri dish within 1 mL dissociation mix before transferring the resulting tissue fragment suspension to a 5 mL Eppendorf tube containing an additional 2 mL of dissociation mix.
[Proceed quickly at room temperature]
?. [Tissue Dissociation]
Incubate for 10 minutes at 37˚C, with rotation at approximately 14 rpm.Tip: Wrap the lid of the Eppendorf tube with Parafilm to prevent any leakage, and 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 at this step 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: Wrap the lid of the Eppendorf tube with Parafilm to prevent any leakage, and 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]
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 10 mL cold HBSS.Tip: If the mixture appears to have a lot of undigested tissue pieces or mucus, which may clog the 70 µm strainer, use a 100 µm strainer instead.
[Wet Ice]
?. [Tissue Dissociation]
Centrifuge at 400 g for 5 minutes in 4˚C pre-cooled centrifuge. This long spin helps to get rid of fat more efficiently than subsequent short spins.
[Centrifugation]
[Centrifuge]
?. [Red Blood Cell Removal]
Report pellet color (e.g., red, pink, white) in the first row of the table at Step 20.
[Wet Ice]
?. [Red Blood Cell Removal]
Carefully transfer supernatant to the 50 mL “Supernatant 1” tube kept on ice, making sure to remove any fat and to not disturb the 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 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 1.7 mL Eppendorf tube kept on ice.Tip: The volume of ACK should be adjusted to the size and color of the pellet and may be increased up to 1 mL if the 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 ACK Lysing Buffer. 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 pellet is pink or red, revealing a significant portion of red blood cells, repeat steps 15-19. For each round of ACK Lysing Buffer treatment, report the ACK Lysing Buffer volume, treatment duration, and pellet color in the table below.Tip: Avoid exceeding three rounds of 1 min ACK Lysing Buffer treatment, as this may result in important loss of cell viability.  ABCD1RepeatACK Lysing Buffer Volume (µL)ACK Lysing Buffer Treatment Duration (min)Pellet Color20NANA345
[if pink or red pellet]
ABCD1RepeatACK Lysing Buffer Volume (µL)ACK Lysing Buffer Treatment Duration (min)Pellet Color20NANA345
?. [Red Blood Cell Removal]
Carefully transfer supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the pellet.
[Wet Ice]
?. [Red Blood Cell Removal]
Resuspend 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 25)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 25)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 25):
?. [[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 in 500 µL cold PBS with 0.4% BSA.
[Wet Ice]
?. [[Optional] Debris and Cell Clumps Removal]
Filter through 35 µm FACS tube cell strainer.Tip: Alternately, 30 µm MACS SmartStrainer can be used to minimize cell loss.
[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 supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the pellet.
[Wet Ice]
?. [[Optional] Debris and Cell Clumps Removal]
Resuspend in 50 µL cold PBS with 0.4% BSA.
[Wet Ice]
?. [[Optional] Debris and Cell Clumps Removal]
Repeat steps 23-24.
[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: