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• Select Differential Expression Analysis from the Analyze Known Genes section of the RNA-seqSeq workflow
• The Differential Expression Analysis dialog offers the choice of analyzing at Gene-,Transcript-, or Exon-level. Select Gene-level 
• Specify the 1/gene_rpkm (RNA-Seq_results.gene.rpkm) spreadsheet from the Spreadsheet drop-down menu (Figure 1)
  
  

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• Select Contrasts... to open the Configure dialog
• For Select Factor/Interaction, Tissue will be the only factor available as it was the only factor included in the ANOVA model in the previous step; if multiple factors were included, they could be selected in the Select Factor/Interaction: drop-down menu. The levels in this factor are listed on the Candidate Level(s) panel on the left side of the dialog
• Left click to select muscle from the Candidate Level(s) panel and move it to the Group 1 panel (renamedmusclerenamed muscle) by selecting Add Contrast Level > in the top half of the dialog. Label 1 will be changed to the subgroup name automatically, but you can also manually specify the label name 
• Select not muscle from the Candidate Level(s) panel and move it to the Group 2 panel (renamed not muscle) 
• The Add Contrast button can now be selected (Figure 3)
• Select OK to return to the ANOVA dialog
  
  

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