TITLE: HTAPP_Depletion of CD45+ cells from ovarian cancer ascites single cell suspensions 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">Many ovarian cancer ascites samples have a high proportion of CD45-expressing cells (>90%) (CD45+) and a low proportion (<10%) of malignant cells. In such cases, depletion of CD45+ immune cells can be used to increase the proportion of malignant cells, as well as other non-immune cell types, without relying on the expression of epithelial or tumor-specific markers.</div><div class = "text-block"><span>For the Human Tumor Atlas Pilot Project (HTAPP) this protocol was used to deplete CD45+ immune cells from single-cell suspensions obtained from ovarian cancer ascites, and is included as a step in the "HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq" protocol, also available in </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>. It is adapted from the CD45 MicroBeads MACS Separation </span><a href="https://www.miltenyibiotec.com/DE-en/shop/comMiltenyiDatasheet/product?productId=390" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocol</span></a><span> from Miltenyi Biotec (Cat No 130-045-801)</span><span style = "font-style:italic;">. </span><span></span></div><div class = "text-block"><span>Description of this protocol and guidance for testing and selecting methods for processing different tumor and sample types can be found in </span><a href="https://doi.org/10.1038/s41591-020-0844-1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Slyper et al</span></a><span>.</span></div></div>

[STEPS]
?. [Quality Control]
Mix 5 µL of red blood cell-free ascites single-cell suspension with 5 µL Trypan blue and load on hemocytometer. The ascites cell suspension can be obtained following the "HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq" protocol.
?. [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.  AB1Quality Control2Number 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 Aggregages or Doublets (%)12Description of debris (if any)
AB1Quality Control2Number 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 Aggregages or Doublets (%)12Description of debris (if any)
Insert Picture:
?. [Magnetic Labeling]
Centrifuge at 580 g for 5 minutes in 4˚C pre-cooled centrifuge.
[Centrifugation]
[Wet Ice]
?. [Magnetic Labeling]
Remove supernatant as completely as possible without disrupting the cell pellet.
[Wet Ice]
?. [Magnetic Labeling]
If cell number is lower than 10 million, resuspend the cell pellet in 80 µL MACS buffer. If cell number is higher than 10 million cells, resuspend the cell pellet in 80 µL MACS buffer for every 10 million cells.
[Wet Ice]
?. [Magnetic Labeling]
Add 20 µL CD45 microbeads per 80 µL MACS buffer.
[Wet Ice]
?. [Magnetic Labeling]
Incubate on ice or in 4°C refrigerator for 15 minutes.
[Labeling]
[Wet Ice]
?. [Magnetic Labeling]
During the incubation, prime an LS column: insert the column into a MidiMACS separator and let 3 mL of MACS buffer flow through the column, collecting it in the 15 mL "Wash" tube. Once the column is primed, discard the 15 mL "Wash" tube and position the 15 mL "CD45 negative fraction" tube under the column.
?. [Magnetic Labeling]
Following incubation (Step 7), resuspend the labeled cell suspension with 900 µL cold MACS buffer per 100 µL of suspension.
[Wet Ice]
?. [Magnetic Labeling]
Spin the suspension at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.
[Centrifugation]
[Centrifuge]
?. [Magnetic Labeling]
Remove supernatant as completely as possible without disrupting the labeled cell pellet.
[Wet Ice]
?. [Magnetic Labeling]
Resuspend the pellet (up to 100 million cells) in 500 µL cold MACS buffer.
[Wet Ice]
?. [Magnetic Separation]
Transfer the labeled cell suspension to the primed LS column on the MidiMACS separator and collect flow-through in the 15 mL "CD45 negative fraction" tube. This CD45 depleted cell suspension can be used in the "HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq" protocol.Tip: Cell recovery may be increased by washing the column up to three times with 3 mL of MACS buffer.
0 Room temperature