Capture-based enrichment techniques have revolutionized genomic analysis of species and populations for which only low-quality or contaminated DNA samples (e.g., ancient DNA, noninvasively collected DNA, environmental DNA) are available. This chapter outlines an optimized laboratory protocol for generating RNA “baits” for genome-wide capture of target DNA from a larger pool of DNA. This method relies on the in vitro transcription of biotinylated RNA baits, which has the dual benefit of eliminating the high cost of synthesizing custom baits and producing a bait set that targets the majority of regions genome-wide. We provide a detailed protocol for the three main steps involved in bait library construction: (1) making a DNA library from a high-quality DNA sample for the organism of interest or a closely related species; (2) using duplex-specific nuclease digestion to reduce the representation of repetitive regions in the DNA library; and (3) performing in vitro transcription of the repetitive region-depleted DNA library to generate biotinylated RNA baits. Where applicable, we include notes and recommendations based on our own experiences.