In silico SETD2 methylation site analysis

1. From any browser, access the phosphositeplus website (www.phosphosite.org)
   

1. Navigate to Downloads > Datasets from PSP

2. Accept the terms and conditions

3. Download the Methylation_site_dataset.gz file

4. Open file using Microsoft Excel

   
   

2. From the main file, duplicate the sheet (rename "Lysine Methylation")

1. Sort MOD_RSD column alphabetically, and keep only rows with K methylation

   
   

3. For KxP methylation sites:

1. Create a new duplicate sheet (rename "KxP methylation")

2. Use the SITE_+/-_7AA column for the searches below (in our spreadsheet, it was column J)

3. Create a column "K?P" --> use function =SEARCH("K?P",J2) and then drag down to create the column for all rows

1. In our spreadsheet, it was column K

2. This will provide all AA sequences that have a KxP motif, with the position of the K indicated as a number
   

4. Sort column "K?P" in ascending order, and keep only rows with values present

5. Create another column "K?P"--> use function =MID(J2,SEARCH("K?P",J2,3) and drag down to create a column for all rows

1. In our spreadsheet, it was column L

2. This will provide all the KxP motifs from the SITE_+/-_7AA column

6. Create another column "k?P" --> use function =FIND("k", L2) and drag down to create a column for all rows

1. In our spreadsheet, it was column M

2. This will provide a value of 1 if the lysine from the KxP is UNcapitalized, i.e., it is the site that is being modified

3. If the motif from column L has a methylated lysine at another spot, it will return 2 or 3

4. If the motif from column L had a CAPITALIZED K, then this column will return #VALUE

7. Create a new duplicate sheet (rename "KxP no repeats")

1. Delete all rows in which the "k?P" column returned #VALUE

2. Sort sheet by GENE column (in our spreadsheet, it was column A)

3. Use Data --> remove duplicate function and select column A




4. For KxxGG methylation sites

1. Create a new duplicate spreadsheet from the "Lysine Methylation" sheet (rename "KxxG methylation")

2. Repeat above steps using "K??G" instead of "K?P", and "k??G" instead of "k?P"

3. Retain the rows in which the "k??G" =FIND("k",L2) function returned the value of 1 

4. Make the sheet "KxxG methylation no repeats"

   
   

5. Make a new sheet (rename "KxP_KxxG")

1. Copy all rows from the "KxP no repeats" and "KxxG no repeats" 

2. Create a new duplicate sheet (rename "KxP_KxxG no repeats")

1. Sort by GENE (column A) 

2. Use Data --> remove duplicate function and select column A

Spreadsheet used in our analysis available upon request once the protocol has been utilized (please contact cheryl.walker@bcm.edu)

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