Depletion of rRNA after RNA-Seq library preparation enriches for RNAs of interest and preserves the diversity of the transcriptome. A, a significantly greater number of small RNAs (<200 bases) is retained using a post–library depletion strategy compared to a pre–library depletion strategy. Libraries were constructed using the SEQuoia Complete Stranded RNA Library Prep Kit (catalog #17005726) and either rRNA depleted (NEBNext rRNA Depletion Kit, New England Biolabs [NEB], #E6350L) Human Placenta Total RNA (Thermo Fisher Scientific Inc., #AM7950) or nondepleted Human Placenta Total RNA. The library constructed using nondepleted total RNA was subsequently depleted using the SEQuoia RiboDepletion Kit. Libraries were then sequenced on a NextSeq^® 500 Sequencing System (Illumina, Inc.) to a read depth of 10 million. The number of unique small RNAs detected (RPKM ≥5) was significantly more in the post–library depletion sample, resulting in a more complex library and richer dataset.