1- Log in into HiPathia. For further information on this step visit Logging in. 2- We will work with a Breast Cancer dataset from the repository The Cancer Genome Atlas. You can download the expression matrix and the experimental design from these links:

• Expression matrix: brca_genes_vals_bn.txt
• Experimental design: brca_normal-basal_ed.txt

3- Upload the data to HiPathia in the data panel by clicking on My data. For further information on this step visit Upload your data. 4- Press the Differential signaling button.

5- In the Input data panel select Expression matrix. Press the File browser of the Expression matrix file, and select the desired file.

6- In the Design data panel select Two group comparison. Press the File browser of the Experimental design file, and select the desired file. Automatically Condition 1 and Condition 2 files are selected. Change Condition 1 to “Tumor” and Condition 2 to “Normal”.

7- Select Human as species (by default). 8- In the Function level analysis, check both checkboxes, Gene ontology and Uniprot keywords. 9- In the Pathways panel select all the pathways (by default). 10- In the Job information panel, press the File browser button and select the desired output folder. In this case we will use analysis. Give a name to the job, for example “BRCA Differential signaling”.

11- Press the Launch job button. A job will be created and listed in the jobs panel. You can access this panel by clicking on the My jobs button.