Abstract
Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s4 U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s4 U-RNA is the efficiency of the chemistry used to enrich s4 U-RNA. Here, we present a protocol for s4 U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s4 U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads. The efficiency of this chemistry reduces enrichment bias and requires less starting material, thereby expanding the utility of s4 U to study RNA biology. © 2016 by John Wiley & Sons, Inc.