Source of background	Experimental strategy
Nonspecific RNA binding to surfaces	• Use low retention tubes • Transfer streptavidin beads or MTS resin to new tube before elution step • Perform nucleoside conversion chemistry after enrichment to identify s4U-containing reads during bioinformatic analysis
Nonspecific RNA binding to beads	• Fragment total RNA before enrichment • Pre-block beads with polyvinylpyrrolidone or glycogen • Add high salt rinses to RNA on beads • Compare s4U-RNA to a non-s4U RNA sample • Perform nucleoside conversion chemistry after enrichment to identify s4U-containing reads during bioinformatic analysis
Nonspecific RNA base-pairing with s4U-RNA on beads	• Add low salt and/or denaturing washes to RNA on beads • Perform nucleoside conversion chemistry after enrichment to identify s4U-containing reads during bioinformatic analysis
Contamination from endogenously thiolated RNAs (e.g. 2-thiouridine-containing tRNA)	• Compare s4U-RNA to a non-s4U RNA sample