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Purpose:
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Based on extrapolation, approximately a quarter of all RAPs had been classified as iRAPs due to their transcription inhibitory potential. To provide a better estimate, we developed a high-throughput cell sorting-based assay to test the in cis activity of many RAPs in parallel. To do so, we first cloned the original SELEX RAP pool with all the RAP sequences in the form of DNA into a GFP-based reporter plasmid (pGFP). We further sorted the bacterial populations with RAP-containing plasmids (pSELEX_RAPs-GFP) to obtain a fraction of cells with reduced GFP expression (i.e., the cells containing an iRAP upstream of the GFP). To avoid false positives, we applied stringent gating resulting in a collected fraction of ~9% of all cells. We deep-sequenced the reporter plasmids from this subgroup and analyzed the output to identify RAPs that cause Rho-dependent termination |
