A library of transcript leaders from S. cerevisiae and S. paradoxus was cloned upstream of a YFP reporter gene to quantify their effects on translation. Mutant versions were designed to remove AUG-initiated uORFs by mutating their start codons to AAG. Yeast transformed with this library were subjected to FACS based on expression of YFP and an internal control mCherry reporter. Transcript leaders were sequenced from bins of sorted cells with different YFP expression values (FACS-uORF). Reporter transcripts were also separated by sucrose gradient fractionation and sequenced to quantify ribosome loading on reporter mRNA.