TITLE: HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq
AUTHORS: Benjamin Izar, Parin Shah, Mei-Ju Su, Isaac Wakiro, Sara Napolitano, Jingyi Wu, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to process human ovarian cancer ascites to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology.</div><div class = "text-block"><span>A majority of ascites samples have a high proportion (>90%) of CD45-expressing (CD45+) cells, thus greatly reducing the ability to capture non-immune and malignant cells. To address this issue, we have included a </span><a href="http://dx.doi.org/10.17504/protocols.io.bhbij2ke" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">CD45+ cell depletion</span></a><span> step to reduce the number of CD45+ cells. In such samples, where the proportion of immune cells is very high, a large proportion of both CD45+ and CD45- cells can be recovered post-depletion, allowing simultaneous assessment of the immune and non-immune microenvironment. In cases with a lower initial proportion of CD45+ cells, however, non-immune cells tend to be dominant following CD45+ cell depletion.</span></div><div class = "text-block">Of note, many ascites samples contain multicellular aggregates, or spheroids, which pose challenges during sample processing and dissociation. Larger aggregates are removed through an initial 70 μm straining (Step 14) whereas smaller residual aggregates, when present, can be removed through a final 30 μm straining (optional Step 20). Yet, we have encountered instances where small aggregates cannot be efficiently removed through straining. This protocol is not optimized for those cases. In addition, we would like to note that ascites can be quite variable and further optimization of this protocol may be needed to handle cases with atypical characteristics. </div><div class = "text-block">For the Human Tumor Atlas Pilot Project (HTAPP), this protocol was successfully applied to 4 ovarian cancer ascites samples. </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>.</span></div></div>

[STEPS]
?. [Sample Description and Allocation]
Report sample processing information.
[Wet Ice]
Sample ID:
Date:Person Processing:
?. [Sample Description and Allocation]
Report the volume and appearance of the sample, including a picture.
[Wet Ice]
Sample Volume (mL):Describe Sample Appearance:
Insert Picture(s) of Sample:
?. [Sample Description and Allocation]
If required, aliquot some of the ascites for different assays and document the allocation process. For single-cell RNA-seq, we recommend starting with at least 200 mL of ascites since a large number of cells will be lost during CD45+ cell depletion. This protocol assumes that ascites have been collected in four 50 mL conical tubes.
[Wet Ice]
Describe Sample Allocation:
?. [Sample Description and Allocation]
Spin down the tubes at 580 g for 5 min in 4°C pre-cooled centrifuge.
[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 11.
[Wet Ice]
?. [Red Blood Cell Removal]
Aspirate off and discard supernatant.
[Wet Ice]
?. [Red Blood Cell Removal]
Resuspend each cell pellet in 1-5 mL cold ACK Lysing Buffer. If cell suspensions are in different tubes, combine them into a smaller number of 50 mL or 15 mL tubes without exceeding one third of the volume of the tubes. For instance, four 5 mL cell suspensions (20 mL in total) can be combined in two 50 mL tubes, and four 1 mL cell suspensions (4 mL in total) can be combined in one 15 mL tube.Tip: The volume of ACK should be adjusted to the size and color of the cell pellets. Combining cell pellets helps minimizing cell loss while also reducing processing time.
[Wet Ice]
?. [Red Blood Cell Removal]
Incubate on ice for 3 minutes.
[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.
[Wet Ice]
?. [Red Blood Cell Removal]
Centrifuge at 580 g for 5 minutes in 4˚C pre-cooled centrifuge.
[Centrifugation]
[Wet Ice]
?. [Red Blood Cell Removal]
If the pellet is pink or red, revealing a significant portion of red blood cells, repeat steps 6-10. 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 3 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]
Aspirate off and discard supernatant.
[Wet Ice]
?. [Red Blood Cell Removal]
Resuspend the cell pellet in 20 mL cold PBS.
[Wet Ice]
?. [Red Blood Cell Removal]
Filter the cell suspension through a 70 µm cell strainer into a 50 mL conical tube. Wash strainer with 20 mL cold PBS to recover as many cells as possible.
[Wet Ice]
?. [CD45+ Cells Depletion]
Deplete CD45+ cells using the protocol below, with the aim to balance the proportion of immune and non-immune cells. This step may be skipped for samples known to already contain a large enough proportion of non-immune cells.
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?. [Quality Control]
Centrifuge at 580 g for 5 minutes in 4˚C pre-cooled centrifuge.
[Centrifugation]
[Centrifuge]
?. [Quality Control]
Discard supernatant and resuspend cell pellet in 50 µL 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 aggregates, 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 Aggregate Removal (Step 20)2Number of Viable Single Cells Counted3Number of Dead Single Cells Counted4Number of Cell Aggregates or Doublets Counted5Concentration of Viable Single Cells (cells/µL)6Concentration of Dead Single Cells (cells/µL)7Concentration of Cell Aggregates 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 Aggregates or Doublets (%)12Description of debris (if any)
ABC1Initial Quality ControlQuality Control after Optional Debris and Cell Aggregate Removal (Step 20)2Number of Viable Single Cells Counted3Number of Dead Single Cells Counted4Number of Cell Aggregates or Doublets Counted5Concentration of Viable Single Cells (cells/µL)6Concentration of Dead Single Cells (cells/µL)7Concentration of Cell Aggregates 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 Aggregates 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 20):
?. [[Optional] Debris and Cell Aggregate Removal]
If the quantity of debris or cell aggregates is too high to load on the 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 aggregates. 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.
[Wet Ice]
?. [[Optional] Debris and Cell Aggregate Removal]
Resuspend in 500 µL cold PBS with 0.4% BSA.
[Wet Ice]
?. [[Optional] Debris and Cell Aggregate Removal]
Filter through 35 µm FACS tube cell strainer.Tip: Alternately, a 30 µm MACS SmartStrainer can be used to minimize cell loss.
[Wet Ice]
?. [[Optional] Debris and Cell Aggregate 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 Aggregate Removal]
Transfer to Eppendorf tube.
[Wet Ice]
?. [[Optional] Debris and Cell Aggregate Removal]
Repeat Steps 16-19.
[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 aggregates lower than 5%.
?. [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: