Sarah Carratt: Week 12
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the of .
In other words, why does the cell react to cold shock by changing the expression of genes associated with these GO terms?'''''
Revision as of 01:59, 12 April 2011
Preparing to Use STEM
- First, I downloaded the software and registered with the website. STEM
- After unziping the file (7-zip > Extract Here), I launched the program using the command window.
- To do this, I went to the start menu and clicked Programs > Accessories > Command Prompt.
- Then I entered the following commands in the window that appeared:
java -mx512M -jar stem.jar -d defaults.txt
Preparing the Spreadsheet
- I opened my master spreadsheet and inserted a new worksheet and named it "stem".
- I copied over data from the "final" worksheet to the "stem" worksheet.
- Renamed the columns: "MasterIndex"→ "SPOT" and "ID"→ "Gene Symbol"
- Deleted all of the data columns except AvgLogFC columns.
- Renamed the data columns with time and units for simplicity.
- 'Save as Text (Tab-delimited) (*.txt).
- Expression Data Info: selected my file (no normalization/add 0 and Spot IDs included in the data file)
- Gene Info: 'Saccharomyces cerevisiae (SGD) with no cross references'and no gene locations
- Options: STEM Clustering Method was selected, no changes
- Execute: Run the program
Viewing and Saving STEM Results
- Changed to "Based on real time" from Interface Options and took a screenshot of this window (saved to powerpoint)
- Opened detailed plots of each profile and took individual screenshots (saved to powerpoint)
- "Profile Gene Table" and "Profile GO Table": saved tables and uploaded to lionshare with correct names
Analyzing and Interpreting STEM Results
- Selected profile 9 for further interpretation, which was mostly down regulated and never up-regulated. I chose this gene because I find down-regulated genes easier to understand and explain. It also followed a simple pattern that I knew I would be able to put into context.
- 221 genes were assigned to this profile.
- 55.9 (56) genes were expected to be assigned to this profile.
- The p value is 1.5E-65 (significant).
- Opened the GO list and selected the third row. From the menu, I clicked Data > Filter > Autofilter.
- Looked at terms with p value of < 0.05: 38 terms
- Looked at corrected values with p value of < 0.05: 2 terms
- GO:0005737 cytoplasm: All of the contents of a cell excluding the plasma membrane and nucleus, but including other subcellular structures.
- GO:0044445 cytosolic part
- GO:0005829 cytosol
- GO:0005622 intracellular
- GO:0044424 intracellular part
- GO:0008483 transaminase activity
- GO:0016769 transferase activity, transferring nitrogenous groups
- GO:0034637 cellular carbohydrate biosynthetic process
- GO:0009063 cellular amino acid catabolic process
- GO:0044444 cytoplasmic part
In other words, why does the cell react to cold shock by changing the expression of genes associated with these GO terms?
- Opened web window .
- Opened gene list in Excel for profile 9.
- Copied the list of gene IDs into web box for ORFs/Genes.
- Checked the box for Check for all TFs.
- Unchecked the box for Indirect Evidence.
- Clicked the Search button.
- Top 10 transcription factors: Ste12p (36.2%), Rap1p (29.9%), Fhl1p (18.6%), Cin5p (14.0%), Phd1p (13.6%), Sok2p (13.1%), Yap6p (12.7%), Yap5p (11.3%), Skn7p (10.4%), Yap1p (9.5%).
- GLN3 is on the list, representing 3.2% and 7 genes: YDR210w, YEL007w, UGA1, CPS1, PUT1, ZEO1, WTM1.
- Transcription factors I used to general the matrix and diagram: CIN5, CUP9, FHL1, GTS1, HSF1, MSN1, MSN4, NRG1, RAP1, RCS1, REB1, ROX1, RPH1, YAP1, YAP6, GLN3, STE12, PHD1, SOK2, YAP5, SKN7
- I added the top five (non-overlapping) transcription factors to the list because I figured that if they represent such a large portion of regulated genes that they should be included in the map.
- Before I generated the figures, I unchecked the box for "Indirect Evidence" and selected "JPEG" from the drop-down menu for the "Output Image".
- Clicked "Generate"
- Saved RegulationMatrix to Lionshare.
- Clicked on the "Image" link to see the diagram of the network.
- Pasted image into my PowerPoint file and uploaded to Lionshare.