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Once we have classified our cells, we can use this information to perform comparisons between cell types or between experimental groups for a cell type. In this project, we only have a single sample, so we will compare cell types.

• Click the Antibody Capture data node
• Click Differential analysis 
  
• Click GSA

The first step is to choose which attributes we want to consider in the statistical test. 

• Check Cell type to include it in the statistical test
• Click Next 

Next, we will set up the comparison we want to make. Here, we will compare the Activated and Mature B cells.

• Check Activated B cells in the top panel
• Check Mature B cells in the bottom panel 
• Click Add comparison

The comparison should appear in the table as Activated B cells vs. Mature B cells.

• Click Finish to run the statistical test (Figure ?)

The GSA task produces a GSA data node.

• Double-click the GSA data node to open the task report

The report lists each feature tested, giving p-value, false discovery rate adjusted p-value (FDR step up), and fold change values for each comparison (Figure ?)

In addition to the listed information, we can access dot and violin plots for each gene or protein from this table.

• Click Image Modified in the CD45RA_TotalSeqB row

This opens a dot plot in a new data viewer session, showing CD45A expression for cells in each of the classifications (Figure ?)

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• Expand the Summary card
• Switch on Violins
• Switch on Overlay
• Switch on Colored
• Expand the Data card
• Use the slider to increase the Jitter
• Expand the Color card
• Use the slider to decrease the Opacity (Figure ?)

• Click the project name to return to the Analyses tab

To visualize all of the proteins at the same time, we can make a hierarchical clustering heat map.

• Click the GSA data node
• Click Exploratory analysis in the toolbox
• Click Hierarchical clustering/heat map
• Check Samples at the top to cluster the cells in addition to features
• Click Finish to run with other default settings
• Double-click the Hierarchical clustering task node to open the heat map (Figure ?)

The heat map can easily be customized to illustrate our results.

• Click Image Added to transpose the heat map
• Set High to 2.6 to match the low range
• Set the Sample dendrogram to By sample attribute Cell type
• Set Attributes to Cell type
• Click Image Added and set Rotation to 0
• Uncheck Samples under Show labels

Differential Analysis, Visualization, and Pathway analysis - Gene Expression Data

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