Overview
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.
Effective depletion of rRNA from libraries constructed using a variety of RNA library prep kits and a broad input range. A, the SEQuoia RiboDepletion Kit effectively removes rRNA fragments from libraries constructed using the NEBNext Ultra II Directional RNA Library Prep Kit (
), Illumina® TruSeq® Stranded Total RNA Library Prep Kit (), or KAPA RNA HyperPrep Kit (Roche Sequencing, ). Each library was constructed using 100 ng of Human Placenta Total RNA according to the manufacturer’s instructions. A broad range of input cDNA (0.1–20 ng) from each library was depleted using the SEQuoia RiboDepletion Kit and sequenced to a read depth of 10 million. On average, <10% of sequencing reads map to rRNA and mt rRNA, indicating efficient and consistent depletion across a broad input range. B, transcriptome profiling is preserved when libraries are depleted in a pooled reaction. The number of total unique transcripts (RPKM >0.1) in the libraries depleted in the pooled reaction was compared to the number in the individually depleted libraries. For each library, the correlation R was >0.97. These data suggest RNA-Seq libraries can be depleted in one multiplexed reaction without compromising depletion efficiency or transcript expression. RPKM, reads per kilobase per million mapped reads; rRNA, ribosomal RNA.
RNA-Seq libraries can be depleted in a 96-plex pooled reaction without affecting library quality. A, similar depletion efficiency is obtained when libraries are depleted individually compared to when they are pooled and depleted in one multiplexed reaction, even if the libraries are constructed using different library preparation kits. Ninety-six individual pre-indexed libraries were constructed using 100 ng Human Placenta Total RNA: 48 libraries were constructed using the SEQuoia Complete Stranded RNA Library Prep Kit and 48 libraries with the NEBNext Ultra II Directional RNA Library Prep Kit. An aliquot of each library was either individually depleted (singleplex) using the SEQuoia RiboDepletion Kit or pooled (multiplex) by mixing an equal molar ratio of the libraries and then depleted. Libraries were sequenced on the NextSeq 500 Sequencing System to a read depth of 3 million reads. Consistently, the percentage of reads mapping to rRNA in a library that was depleted in the multiplex reaction was comparable to those libraries that were depleted individually.
