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By default, each sample in a multi-sample data set is plotted on its own t-SNE. 

• Select the Single cell data Click the Filtered counts node
• Select t-SNE from the Visualizations Exploratory analysis section of the task menu (Figure 1)
  
  

• Select Click Finish from the t-SNE dialog to run t-SNE with the default settings

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

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

The t-SNE plot will open to the first sample in the data set, Astrocytoma 1 (Figure 4). 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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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 goal of this experiment 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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The cells will turn black and a text box Gene ID will open below the drop-down box. 

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The cells will be colored from black to green based on their expression level of CD14, with cells expressing higher levels more green (Figure 8). CD14 is a known marker for microglia and macrophage cells, used by the authors of the original study to classify microglia/macrophage cells. 

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In Partek Flow, we can color cells with up to three genes at a time. We will now add a second gene, MOBP. 

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Relative expression of the two genes for selected cells can be visualized on the legend. 

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Selected cells are shown in bold and unselected cells are dimmed.

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Numerical expression levels for each gene can be viewed for individual cells. 

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Now that cells are colored by expression of a microglia/macrophage marker, CD14, and a oligodendrocyte marker, MOBP, we can classify any cell that does not fall into one of these two groups as malignant 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 or MOBP-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. 

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