catcheR_design - Oligonucleotide Design

This step complements Supplemental Protocol 1.

1. In a new working folder, prepare the following files:

   1. A comma-separated values (CSV) file with three columns listing: (1) the forward oligos from the TRC shRNA library (2) the corresponding barcodes (BC) (3) the shRNA names

      Below is an example for the shRNA described in FigureSPOneOne:

   CCGGCAAGTACTCCTTGCTGGATTGCTCGAGCAATCCAGCAAGGAGTACTTGTTTTTG,CAGTTCCA,SMAD2.1
   ...
   

   2. (Optional) A .txt file with a newline-separated list of 5’-3’ restriction sites, or other sequences to be avoided in the shRNAs. By default these are SalI, SwaI, and AscI:

   GTCGAC
   ATTTAAAT
   GGCGCGCC
   

2. Run catcheR_design:

   catcheR_design(
       group = c("docker", "sudo"),
       folder,
       sequences,
       gibson.five = "AGTTCCCTATCAGTGATAGAGATCCC",
       gibson.three = "GTAGCTCGCTGATCAGC",
       fixed = "GTCGACATTTAAATGGCGCGCC",
       restriction.sites = NULL
   )
   

   `catcheR_design` arguments:

   1. group: string, one of “docker” or “sudo” depending on user permissions (For a detailed explanation of Docker user groups, see: https://docs.docker.com/engine/install/linux-postinstall/ *)

   2. folder: string with the path to the working folder

   3. sequences: string with the CSV file name from step 1a

   4. gibson.five: (optional) string with the 5’ Gibson homology sequence

   5. gibson.three: (optional) string with the 3’ Gibson homology sequence

   6. fixed: (optional) string with the multicloning site

   7. restriction.sites: (optional) string with the .txt file name from step 1b

   Example usage:

   catcheR_design(
       group = "docker",
       folder = "path/to/folder",
       sequences = "filename.csv",
       restriction.sites = "filename.txt"
   )
   

   `catcheR_design` outputs:

   1. output.txt – contains the oligo sequences to be used for synthesis (see FigureSPOneOne)

   2. bad_oligos.txt – lists the shRNAs containing forbidden restriction sites (highlighted in lowercase characters). This can be used to refine the shRNA list.

   Example output from bad_oligos.txt:

   AGTTCCCTATCAGTGATAGAGATCCCGGACATAATCACTGCGTAATCCTCagatctTACGCAGTGATTATGTCCTTTTTTTGT-
   CGACATTTAAATGGCGCGCCNNNNNNGCTGAAGAGTAGCTCGCTGATCAGC,GATA4
   ...