TITLE: HTAPP_Depletion of CD45+ cells from single-cell suspension for single-cell RNA-seq
AUTHORS: Isaac Wakiro, Sara Napolitano, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In some tumor specimens the proportion of malignant cells is relatively low and the that of immune cells high. In these cases, we considered strategies to deplete CD45+ immune cells as a way to both enrich for epithelial cells without using specific epithelial cells markers, and to maintain the stromal cells.  </div><div class = "text-block">The protocol described here is used for the depletion of CD45+ expressing immune cells from human tumor-derived single-cell suspensions. It is adapted from the CD45 MicroBeads MACS Separation protocol from Miltenyi Biotec. </div><div class = "text-block">For the Human Tumor Atlas Pilot Project (HTAPP), CD45 depletion was used to increase the recovery of malignant epithelial cells from lung non-small cell adenocarcinoma samples in which the proportion of these types of cells was low.</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://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>

[STEPS]
?. [Initial Quality Control]
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on hemocytometer.
?. [Initial Quality Control]
Count and record the number of viable single-cells, dead single-cells, cell doublets, and whether debris are present, then calculate additional quality control metrics below. Take picture if possible.  AB1Initial Cell Suspension2Number 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)
AB1Initial Cell Suspension2Number 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:
?. [Magnetic Labeling]
Centrifuge cell suspension in a 1.5 mL Eppendorf tube at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.
Centrifuge: 500 34, 4 min, 4 10
?. [Magnetic Labeling]
Carefully transfer supernatant to the 15 mL “Supernatant” tube kept on ice without disturbing the pellet.
on ice
?. [Magnetic Labeling]
If the cell pellet contains less than 107 cells,  resuspend in 80 µL MACS buffer. If the cell pellet contains more than 107 cells, resuspend in 80 µL MACS buffer per 107 cells.
on ice
Volume of MACS Buffer Used (µL):
?. [Magnetic Labeling]
Add 20 µL CD45 microbeads per 80 µL MACS buffer.
on ice
Volume of Microbeads Used (µL):
?. [Magnetic Labeling]
Incubate in 4°C refrigerator for 15 minutes.
[Labeling]
[Refrigerator]
?. [Magnetic Labeling]
During the incubation, insert an MS column into the MiniMACS separator set on the MACS MultiStand. Prime the column with 500 µL MACS buffer, collecting the effluent in the 15 mL "Wash" tube.
0 Room temperature
?. [Magnetic Labeling]
Following incubation (Step 7), add 1 mL cold MACS buffer per 100 µL suspension.
on ice
Volume of MACS Buffer Used (µL):
?. [Magnetic Labeling]
Centrifuge suspension at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.
Centrifuge: 500 34, 4 min, 4 10
?. [Magnetic Labeling]
Carefully transfer supernatant to the 15 mL “Supernatant” tube kept on ice without disturbing the pellet.
on ice
?. [Magnetic Labeling]
If the cell pellet contains less than 108 cells,  resuspend in 500 µL MACS buffer. If the cell pellet contains more than 108 cells, resuspend in 500 µL MACS buffer per 108 cells.
on ice
Volume of MACS Buffer Used (µL):
?. [Collection of the CD45- fraction]
Position the 15 mL "CD45-" tube under the MS column, discarding the "Wash" tube.
0 Room temperature
?. [Collection of the CD45- fraction]
Transfer cell suspension (up to 2x108 cells) to the primed MS column on the MiniMACS separator and collect the flow-through in the 15 mL "CD45-" tube.
0 Room temperature
?. [Collection of the CD45- fraction]
Wash the MS column by adding 500 µL MACS buffer and collecting the flow-through in the 15 mL "CD45-" tube. Repeat wash two more times.
0 Room temperature
?. [Collection of the CD45- fraction]
Transfer the "CD45-" tube on ice.
on ice
?. [[Optional] Collection of the CD45+ fraction]
Remove the MS column from the MiniMACS separator and position it on top of the 15 mL "CD45+" tube.
0 Room temperature
?. [[Optional] Collection of the CD45+ fraction]
Add 1 mL of MACS Buffer to the column and immediately elute the CD45+ cells by firmly pushing the plunger into the column.
0 Room temperature
?. [[Optional] Collection of the CD45+ fraction]
Transfer the "CD45+" tube on ice.
on ice
?. [Cell Concentration]
Transfer the CD45- fraction to two 1.5 mL "CD45-" Eppendorf tubes on ice. (If a CD45+ fraction has been collected, transfer it to one 1.5 mL "CD45+" Eppendorf tube on ice.)
on ice
?. [Cell Concentration]
Centrifuge cell suspensions at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.Tip: If the number of cells is expected to be small (e.g., less than 50,000 cells), higher recovery may be obtained by centrifugation for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to but not exceeding 11,000 g (do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death).
Centrifuge: 500 34, 4 min, 4 10
?. [Cell Concentration]
Carefully transfer supernatant from the "CD45-" tubes to the 15 mL “CD45- Supernatant” tube kept on ice without disturbing the pellet. (If a CD45+ fraction has been collected, transfer the supernatant from the "CD45+" tube to the 15 mL "CD45+ Supernatant" tube kept on ice, without disturbing the pellet.)
on ice
?. [Cell Concentration]
Resuspend and combine both CD45- cell pellets in a total volume of 50 µL cold PBS with 0.4% BSA. (If a CD45+ fraction has been collected, resuspend the CD45+ pellet in 50 µL cold PBS with 0.4% BSA.)
on ice
?. [Final Quality Control]
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on hemocytometer.
?. [Final 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.  ABC1CD45- FractionCD45+ Fraction (if collected)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)
ABC1CD45- FractionCD45+ Fraction (if collected)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 CD45- fraction:Insert Picture for CD45+ fraction (if collected):
?. [Loading on 10x]
If necessary, adjust the concentration of the CD45- fraction 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%.
on ice
?. [Loading on 10x]
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: