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).

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).

Globin Depletion Increases Gene Detection

RiboCop for Human/Mouse/Rat plus Globin (HMR+Globin) simultaneously depletes rRNA and globin mRNA, providing a highly convenient workflow for blood samples and freeing up sequencing space for RNAs of interest (Fig. 1). Thus, removal of globin mRNA leads to a significant increase in gene detection (Fig. 2).

Figure 2 | Increased gene detection upon globin depletion. A) The number of detected genes per number of reads uniquely mapping to exons (FeatureCounts) was plotted for the samples shown in Figure 1. B) Bar plot illustrating the number of detected genes for the samples shown in Figure 1 at 1 M reads /sample, normalized to Counts Per Million (CPM) at a threshold of CPM > 1.

Figure 2 | RiboCop maintains unbiased expression profiles while efficiently removing undesired ribosomal RNA. Correlation of transcript abundance in untreated samples vs. samples depleted with RiboCop for Gram Negative Bacteria for 1 ng – 1 µg E. coli RNA input. Libraries were prepared using Lexogen’s CORALL Total RNA-Seq Library Prep Kit and sequenced on Illumina NextSeq (1×75 bp). Reads were mapped against the MG1655 reference genome using STAR aligner and counted with FeatureCounts (including multi mappers).

Figure 4 | RiboCop META maintains constant transcript abundance across various species. Correlation of transcript abundance in untreated samples vs. samples depleted with RiboCop for Mixed Bacterial Samples (META) for 100 ng RNA input of three different species. Library preparation, sequencing, and data analysis were performed as described in Fig. 2. Reads were mapped against the E. coli MG1655, P. aeruginosa PA01, or B. subtilis 168 reference genomes, respectively.

Excellent Reproducibility and Depletion Without Off-target Effects

RiboCop protocols are robust and highly reproducible. Correlation plots show excellent reproducibility between replicates (Fig. 5) independent of the species or the Probe Mix used for depletion.

Figures 2 and 4 additionally show a high correlation between transcript abundance in untreated vs. ribo-depleted samples for a wide input range (1 ng to 1 µg, Fig. 2) as well as across various species (Fig. 4) without outliers. This indicates that depletion with RiboCop for Bacteria occurs without of off-target effects.

Figure 5 | Excellent reproducibility between replicates. Correlation of replicates for two independent depletion reactions using 10 ng total RNA of the indicated species. Total RNA was ribo-depleted with RiboCop for Gram Negative or Gram Positive Bacteria, respectively. Library preparation, sequencing, and data analysis were performed as described in Fig. 4.

RiboCop for Bacteria Selection Tool

If you are uncertain which kit to choose, then the RiboCop for Bacteria Kit Selection Tool can help to find the best-suited kit for your species of interest.