title: FACS-uORF-TotalLib-R3
design: Plasmid DNA libraries were prepared by three rounds of PCR.  8 cycles of PCR were performed in a 20 l reaction using 10 ng of plasmid library template with primers to the ENO2 promotor (Eno2_lib_F1) and YFP sequences (FACs-uORF-YFP-R). The PCR reaction was purified using AMPure XP beads. A second reaction of 6 cycles PCR cycles used primers that included random bases to stagger sequencing reads (FuORF_2_DNA_N0-N7_F, and FuORF_2_DNA_N0,2,6_R).  The second PCR was purified using AMPure XP beads and used as a template for a third round of 15 PCR cycles to incorporate Illumina sequences and barcodes, using the primers RPF_F and RPF_R.  All PCR amplifications were performed using Q5 DNA polymerase.