...

• Name the classification Malignant 
• Select Save (Figure 15)
  

...

Plotting samples together on t-SNE

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 microglia/macrophage and oligodendrocyte cell types. 

• Select the Single cell data data node
• Select t-SNE from the Visualizations section of the task menu
• Select Configure on the t-SNE dialog (Figure 21)
  
  

• Deselect the Split cells by sample option under Misc
• Select Apply (Figure 22)
  
  

•  Select Finish to run the t-SNE task

The t-SNE task will be added as a new green layer in the analysis tab (Figure 23).

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 24).

• 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 25).

Using the known maker genes, CD14 and MOBP, 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 CD14 in the new Gene ID box
• Select CD14 from the list of genes in the data set
• Select the Image Added icon next to CD14
• Type MOBP in the new Gene ID box
• Select MOBP from the list of genes in the data set

After coloring by CD14, a microglia/macrophage marker, and MOBP, a oligodendrocyte maker, these two cell populations are clearly visible (Figure 26). 

• Activate the 3D lasso tool by selecting Image Added
• Draw the lasso around the cluster of red cells and click the circle to close the lasso
• Select Save classifications

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

• Name the classification Oligodendrocytes
• Select Save
  

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

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

Red CD14 positive cells are the microglia/macrophages from every sample. 

• Activate the 3D lasso tool by selecting Image Added
• Draw the lasso around the cluster of green cells and click the circle to close the lasso
• Select Save classifications
• Name the classification Microglia
• Select Save
• Switch modes by selecting Image Added
• Deselect by clicking on any black space on the plot

Finally, we will classify all unclassified cells on the plot as the malignant 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 Added
• Draw the lasso around the grey cells and click the circle to close the lasso
• Select Save classifications 
• Name the classification Malignant
• Select Save

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

• Select Save classifications
• Select Save when asked to confirm

The pipeline view will open and the Classify cells tasks will run, generating a new green-layer Classified groups data node (Figure 27).