Fig. 1.

Experimental design. GFP reporter expression was driven by the EF-1α promoter and potentially modulated by a variable 8mer inserted into the human IQGAP1 3′UTR. The 5′UTR of the GFP reporter contains an intron. Expression of dsRed was used to control for transcriptional noise at the reporter locus, and was driven by the PGK promoter. The flippase recombination target (FRT) site allows this plasmid to undergo site-specific recombination in HEK293-TRex-FLP cells, such that only cells that integrate this construct at the intended locus via the FRT site gain hygromycin resistance. After integration, cells with normal transcriptional activity at the reporter locus (as determined by dsRed levels), and that are potentially undergoing differential post-transcriptional regulation (as determined by GFP levels), were isolated via fluorescence activated cell sorting (FACS). From FACS isolated sub-populations, the portion of the 3′UTR containing the variable 8mer was PCR amplified, thereby adding Illumina adapter sequences, and allowing 8mers in each sorted population to be identified and quantified by Illumina sequencing