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For some data sets, cell types can be distinguished when all samples can be visualized together on one t-SNE plot. We will use a t-SNE plot of all samples to classify glioma, microglia/macrophage , and oligodendrocyte cell types. 

• Select the Single cell data  Filtered counts data node
• Select t-SNE from the Exploratory anlaysis section of the task menu
• Select Configure on the t-SNE dialog (Figure 2119)
  
  

• Click the Split cells by sample option under Misc to uncheck it
• Click Apply (Figure 2220)
  
  

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The t-SNE task will be added as a green layer in the analysis tab (Figure 2321). Layers are created in Partek Flow when the same task is run on the same data node. 

Once the task has completed, we can view the plot.

• Select the green t-SNE plot task node
• Select Task Report from the task menu

In the multi-sample t-SNE plot, each cell is initially colored by its sample (Figure 2422).

• Select 2D from the Plot style section

Viewing the 2D t-SNE plot, while most cells cluster by sample, there are a few clusters with cells from multiple samples (Figure 2523).

Using the known maker genes, BCAN (glioma), CD14 and MOBP(microglia), and MAG (oligodendrocytes), we can assess whether these multi-sample clusters belong to our known cell types. 

• Select Gene expression from the Color by drop-down menu
• Type BCAN in the new Gene ID box
• Select BCAN from the list of genes in the data set
• Select the Image Added icon next to BCAN
• Type CD14 in the new Gene ID box
• Select CD14 from the list of genes in the data set
• Select the  icon next to CD14
• Type MOBP MAG in the new Gene ID box
• Select MOBP MAG from the list of genes in the data set

After coloring by CD14, a microglia/macrophage marker, and MOBP, a oligodendrocyte maker, these two these marker genes, three cell populations are clearly visible (Figure 2624). 

• Activate the 3D lasso tool by selecting 
• Draw the lasso around the cluster of red cells and click the circle to close the lasso (Figure 2725)
  
  

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• Click Classify selection

These red cells are MOBP CD14 positive, indicating that they are the oligodendrocytes microglia from every sample. 

• Name the classification OligodendrocytesMicroglia
• Select Click Save
  

To clearly see the CD14 positive MAG expressing population, clear the current selection.

• Switch modes by selecting Image Removed
• Deselect by double clicking on any black space on the plot

Green CD14 positive Blue MAG expressing cells are the microglia/macrophages oligodendrocytes from every sample. 

• Activate the 3D lasso tool by selecting Image Removed
• Draw the lasso around the cluster of green blue cells and click the circle to close the lasso (Figure 2826)
  
  

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• Click Classify selection
• Name the classification Microglia
• Select Click Save
• Switch modes by selecting Image Removed
• Deselect by double clicking on any black space on the plot

Finally, we will classify all unclassified the BCAN expressing cells on the plot as the malignant glioma cells from every sample.

• Select Classifications from the Color by drop-down menu

Cells are now colored by classification, with Oligodendrocytes in blue, Microglia in red, and unclassified cells in grey.

• Activate the 3D lasso tool by selecting Image Removed
• Draw the lasso around the grey green cells and click the circle to close the lasso (Figure 2927)
  
  

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• Click Classify selection 
• Name the classification MalignantGlioma
• Select Click Save

With every cell from every sample classified, we can proceed to save classifications. 

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view the number of cells classified into each cell type for each sample on the classification summary page. 

• Click Summary 

The fraction of cells of each cell type in each sample is highly heterogeneous (Figure 28). 

• Click Apply classifications 
• Click Apply to confirm classification

The pipeline view will open and the Classify cells tasks will run, generating a new green-layer Classified groups data node (Figure 3029). 

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