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SubtitleText Invoking t-SNE from the task menu AnchorName Invoking t-SNE
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• Click Finish from the t-SNE dialog to run t-SNE with the default settings

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SubtitleText t-SNE task node AnchorName t-SNE task node
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Once the t-SNE task has completed, we can view the t-SNE plot.

• Click the t-SNE node
• Click Task report from the task menu (Figure 3) or double click the t-SNE node

 

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SubtitleText Opening the t-SNE plot AnchorName Opening the t-SNE plot
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The t-SNE plot will open to the first sample in the data set, Astrocytoma 1 MGH36 (Figure 43). Please note that the appearance of the t-SNE plot will differ each time it is drawn so your t-SNE plots will look different than those shown in this tutorial; however, the cell-to-cell relationships indicated will be the same. 

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SubtitleText Viewing t-SNE plot of Astrocytoma 1a single sample AnchorName Viewing single-sample t-SNE
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The t-SNE plot is in 3D by default. You can rotate the 3D plot by lef-clicking and dragging your mouse. You can zoom in and out using your mouse wheel. The 2D t-SNE is also calculated and you can switch between the 2D and 3D plots using the Plot style radio buttons. 

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The t-SNE plot has switched to show the next sample, Astrocytoma 2 MGH42 (Figure 54). 

 

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SubtitleText Viewing t-SNE plot of Astrocytoma 2MGH42 AnchorName Viewing single-sample t-SNE (2)
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The goal of this experiment analysis is to compare malignant cells from two different glioma subtypes, astrocytoma and oligodendroglioma. To do this, we need to identify which cells are the malignant cells we want to include and which cell are the normal cells we want to exclude. 

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• Open the Color by drop-down menu
• Select Gene expression from the drop-down menu (Figure 65)
  
  

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SubtitleText Selecting color by gene expression AnchorName Selecting color by gene expression
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The cells will turn black and a text box Gene ID will open below the drop-down box. 

• Type CD14 Type BCAN in the Gene ID text box
• Select CD14 BCAN from the list of genes in the data set (Figure 76)
  
  

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SubtitleText Coloring cells by CD14 BCAN expression AnchorName Selecting gene
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The cells will be colored from black to green based on their expression level of CD14BCAN, with cells expressing higher levels more green (Figure 87). CD14 is a known marker for microglia and macrophage cells, used by the authors of the original study to classify microglia/macrophage cellsBCAN is highly expressed in glioma cells. 

 

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SubtitleText Cells colored by CD14 expression AnchorName Colored by CD14
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In Partek Flow, we can color cells with up to three genes at a timemore than one gene. We will now add a second glioma marker gene, MOBPGPM6A. 

• Select the  icon next to CD14BCAN
• Type MOBP GPM6A in the new Gene ID box
• Select MOBP GPM6A from the list of genes in the data set

Cells expressing MOBP GPM6A are now colored red and cells expressing CD14 BCAN are colored green. Cells expressing both genes are colored yellow, while cells expressing neither are colored black (Figure 98).

 

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SubtitleText Coloring cells by MOBP BCAN and CD14GPM6A AnchorName Coloring by two genes
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Relative expression of the two genes for selected cells can be visualized on the legend. 

• Activate the 3D lasso tool by selecting  
• Draw a circle around the cluster of red yellow cells (Figure 109)
  
  

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SubtitleText Selecting a group of cells using the 3D lasso tool AnchorName Selecting a group of cells
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Selected cells are shown in bold and unselected cells are dimmed.

The relative expression of the two genes for the selected cells will be shown on the legend as dots (Figure 1110). 

 

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SubtitleText Viewing expression levels for a group of cells AnchorName Viewing expression levels for a group of cells
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Numerical expression levels for each gene can be viewed for individual cells. 

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The expression level for that cell is displayed on the legend for each gene (Figure 1211). 

• Deselect the cell by clicking on any black space on the plot

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SubtitleText Viewing expression levels for an individual cell. The dots on the legend indicate the expression level of the selected cell. AnchorName Viewing expression levels for a cell
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Now that cells are colored by expression of a microglia/macrophage marker, CD14, and a oligodendrocyte marker, MOBP, two glioma cell markers, we can classify any cell that does not fall into one of these two groups as malignant expresses these genes as glioma cells. Because t-SNE groups cells that are similar across the high-dimensional gene expression data, we will consider cells that form a group with CD14 BCAN or MOBPGPM6A-expressing cells as same cell type, even if they do not express the marker gene.

• Click anywhere on the t-SNE plot without a cell to clear the selection

Starting with the Astrocytoma 2 sample, we can classify the malignant cells in each sample. 

• Activate the 3D lasso tool by selecting 
• Draw the lasso around the cluster of black yellow cells and click the circle to close the lasso (Figure 13). You may need to switch to selection mode and rotate the 3D plot to select only cells from the black yellow cluster

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SubtitleText Selecting malignant cells AnchorName Selecting malignant cells
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The number of selected cells is indicated in the Selection section of the menu.

• Select Classify selection (Figure 1412)
  
  

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SubtitleText Classifying selected cells AnchorName Classifying cells
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A dialog to give the classification a name will appear. 

• Name the classification Malignant Glioma
• Select Save (Figure 1513)
  
  

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SubtitleText Classifying selection AnchorName Classifying cells
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Once cells have been classified, the classification is added to the Classifications section of the panel. The number of cells belonging to the classiciation is listed; in Astrocytoma 2MGH42, there are 253 malignant 413 glioma cells (Figure 1514). 

 

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SubtitleText The number of cells in each classification is displayed in the classification section. AnchorName Viewing classifications
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Classifications made on the t-SNE plot are retained as a draft until after you exit the t-SNE task report. The Save classifications button runs a task, Classify cells, which generates a new Classified cells data node. In this tutorial, we will classify malignant cells for each sample before we save the classifications, but if necissary, you can run Classify cells for one sample, exit the t-SNE task report , and continue classifying the next sample later starting with the Classified cells data node. 

• Select Next to move to the next sample, Astrocytoma 3 MGH45
• Rotate the 3D t-SNE plot to allow you to select only cells from the black yellow cluster
• Activate the 3D lasso tool by selecting 
• Draw the lasso around the cluster of black cells and click the circle to close the lasso (Figure 1614). 
  
  

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SubtitleText Classifying malignant cells AnchorName Classifying cells
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• Select Classify selection 
• Type Malignant Glioma or select Malignant Glioma from the prompt (Figure 1715)
• Select Save
  
  

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SubtitleText Adding cells in a second sample to an existing classification AnchorName Classifying cells as an existing classification
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• Repeat these steps for each of the 6 astrocytoma and 4 oligodendroglioma remaining samples

Once all samples have been classified, it is useful to check the number of cells in each sample assigned to each classification. 

• Select Summary (Figure 1816)
  
  

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SubtitleText Navigating to the classification summary AnchorName Navigating to classification summary
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The classifications summary lists every sample, the number of cells in the sample, the number of cells in each classification, and the percentage of cells in each sample that belong to each classification (Figure 1917).

 

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SubtitleText Viewing the classification summary AnchorName Classification summary
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With the malignant cells in every sample classified, it is time to save the classifications.

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The pipeline view will open and the Classify cells tasks will run, generating a Classified groups data node (Figure 2018).

 

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SubtitleText The Classify cells tasks generates a Classified groups data node AnchorName Classify cells task
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One multi-sample t-SNE plot

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