Skip to main content
. 2014 Aug 28;10(8):748. doi: 10.15252/msb.20145136

Figure 1. Analysis of TIS sequences via FACS-seq.

Figure 1

  1. A TIS reporter library was created by degenerate PCR to create a random permutation of the six bases upstream and two bases downstream of a GFP start codon, resulting in a library size of 65,536 TIS sequences. An IRES followed by RFP was used to normalize the GFP expression.
  2. Stably transduced cells were sorted based on translation efficiency Analysis of TIS sequences via FACS-seq (GFP/RFP). 20 gates were drawn such that each gate contained 5% of the total library population. Cells that were off-scale were collected in the last gate.
  3. The TIS sequences from each sorted population were PCR amplified and barcoded.
  4. The barcoded TIS library was pooled and sequenced. The number of reads for each TIS sequence and barcode combination was counted.
  5. An individual histogram for each TIS sequence is created in silico.
Data information: green fluorescent protein, GFP; red fluorescent protein, RFP; internal ribosomal entry site, IRES; 2A slippage site, 2A; puromycin resistance gene, PuroR.