RiboCop for Human/Mouse/Rat V2

Total RNA from mammalian species is comprised of large amounts of undesired ribosomal RNA (rRNA) which accounts for ~80 – 90 % of all RNA. Lexogen’s RiboCop rRNA Depletion Kits for Human/Mouse/Rat remove undesired cytoplasmic (28S, 18S, 5.8S, and 45S rRNA) and mitochondrial rRNA (mt16S, mt12S, and 5S) from intact as well as degraded material, including formalin-fixed, paraffin-embedded (FFPE) samples.

RiboCop for Human/Mouse/Rat (HMR) V2 is offered as a stand-alone (Cat. No. 144) or as a bundled version with the CORALL Total RNA-Seq Library Prep Kit (Cat. No. 146). RiboCop Kits are compatible with all random primed total RNA library prep kits.

Robust Performance Over a Wide Range of Input Amounts

Ribosomal RNA was depleted from Universal Human Reference RNA (UHRR) using RiboCop for Human/Mouse/Rat (HMR) V2 over a wide range of input amounts (1 ng to 1 µg). RiboCop HMR V2 efficiently reduces rRNA reads to below 1% (Fig. 1) while maintaining unbiased transcript abundance (Fig. 2).

Fig_01 - RiboCop HMR V2

Figure 1 | RiboCop rRNA Depletion for Human/Mouse/Rat efficiently removes rRNA across a wide range of input amounts. NGS libraries were prepared using Lexogen’s CORALL Total RNA-Seq Library Prep Kit. Successful depletion was monitored by sequencing (NextSeq500, 1×75 bp) and subsequent analysis of remaining rRNA reads from untreated (Total RNA) and depleted UHRR (1, 100 ng, and 1 µg). The percentage of reads mapping to rRNA is plotted in blue.

Fig_02 - RiboCop HMR V2

Figure 2 | RiboCop maintains unbiased expression profiles while efficiently removing undesired ribosomal RNA from UHRR. Correlation of transcript abundance in untreated samples vs. samples depleted with RiboCop for Human/Mouse/Rat (HMR) V2 for 1 ng – 1 µg UHRR. Libraries were prepared and sequenced as described in Figure 1. Reads were mapped against the GRCh38.95 reference genome using STAR aligner and counted with FeatureCounts (including multi mappers).

RiboCop Performance Across Species

RiboCop enriches RNAs of interest across a wide range of total RNA input amounts from human, mouse, and rat samples (Fig. 3), and shows excellent reproducibility between replicates (Fig. 4).

Fig_03 - RiboCop HMR V2

Figure 3 | RiboCop rRNA Depletion for Human/Mouse/Rat (HMR) V2 removes rRNA from human (UHR), mouse (liver), and rat (liver). Two RNA input amounts of the indicated species were depleted with RiboCop HMR V2. Library preparation and sequencing were performed as described in Figure 1. Reads were mapped against the respective reference genomes for human (GRCh38.95), mouse (mmu_GRCm38.95), and rat (rno_Rnor_6.0.95). The percentage of reads mapping to rRNA is plotted in blue.

Fig_04 - RiboCop HMR V2

Figure 4 | Excellent reproducibility between replicates. Correlation of replicates for two independent depletion reactions using 1 ng, 100 ng, and 1 µg total UHRR as input. Total RNA was ribo-depleted with RiboCop HMR V2. Library preparation, sequencing, and data analysis were performed as described in Figure 2. Reads were mapped against the GRCh38.95 reference genome.

Excellent Reproducibility and Depletion Without Off-target Effects

RiboCop protocols are robust and highly reproducible. Correlation plots show consistent results over a broad range of inputs (Fig. 5). Additionally, Figure 2 shows a high correlation between transcript abundance in untreated vs. ribo-depleted samples for a wide input range (1 ng to 1 µg) indicating that depletion with RiboCop HMR V2 does not affect the abundance of non-targeted transcripts.

Figure 5 | RiboCop HMR V2 maintains constant transcript abundance across various input amounts. Correlation of transcript abundance in samples depleted with RiboCop HMR V2 for 100 ng RNA vs. 1 ng RNA input for human (UHRR), mouse (mouse liver RNA), and rat (rat liver RNA) samples. Library preparation, sequencing, and data analysis were performed as described in Figure 2. Reads were mapped against the respective reference genomes for human (GRCh38.95), mouse (mmu_GRCm38.95), and rat (rno_Rnor_6.0.95), respectively.