Ribo Zero Plus rRNA Depletion Kit FAQs

Which rRNAs are targeted by Ribo-Zero Plus? Ribo-Zero Plus depletes human globin transcripts and rRNAs from multiple species including human, mouse, rat, and bacteria (E. coli and B. subtilis). Illumina cannot guarantee rRNA removal of species outside of the following:

Depletion target	rRNAs targeted
Human Cytoplasmic rRNAs	28S, 18S, 5.8S, 5S
Human Mitochondrial rRNAs	12S, 16S
Human Beta Globin transcripts	HBA1, HBA2, HBB, HBG1, HBG2
Mouse and Rat rRNA	16S, 28S
Gram(-) Bacterial rRNAs	E.coli: 5S, 16S, 23S
Gram(+) Bacterial rRNAs	B. subtilis: 5S, 16S, 23S

Is there a BaseSpace public dataset for Ribo-Zero Plus? Yes. BaseSpace public datasets are found under the "Demo Data" section. Search for "Illumina Stranded Total" to find relevant datasets.

How does Ribo-Zero Plus compare with the legacy Ribo-Zero bacteria depletion kit? Illumina has tested and demonstrated effective rRNA depletion with the Ribo-Zero Plus kit of E. coli (gram-negative) and B. subtilis (gram-positive) bacteria samples only. rRNA depletion of other bacterial samples has not been tested and performance may vary. While Ribo-Zero Plus targets the same bacterial transcript regions as legacy Ribo-Zero bacteria, Ribo-Zero Plus leverages a more precise targeting mechanism than Ribo-Zero legacy. Because of this, Ribo-Zero Plus may sometimes be less tolerant to bacterial species that are more highly divergent in sequence from E. coli and B. subtilis.

How does the new Ribo-Zero Plus differ from legacy Ribo-Zero? Ribo-Zero Plus leverages a different depletion mechanism compared to legacy Ribo-Zero. Ribo-Zero Plus uses DNA probes and enzymatic depletion to remove abundant transcripts from total RNA samples. Legacy Ribo-Zero leverages biotinylated probes to pull down abundant transcripts via streptavidin beads. When paired with the Illumina Stranded Total RNA prep, Ribo-Zero Plus offers key advantages that enable enhanced study flexibility. These advantages include a one tube (single kit) to deplete multiple species functionality, enhanced depletion at lower total RNA input amounts, and a more precise mechanism for depletion of targeted transcripts.

Where is the reference guide for Ribo-Zero depletion? Ribo-Zero Plus can be purchased either as a standalone kit, or bundled with Illumina Stranded Total. If using Ribo-Zero Plus standalone or with the Illumina Stranded Total library preparation kit, follow the Illumina Stranded Total with Ribo-Zero Plus reference guide.

If using standalone Ribo-Zero Plus with TruSeq Stranded Total library preparation, follow the reference guide for TruSeq Stranded Total with Ribo-Zero Plus. Note that when used with TruSeq Stranded Total, Ribo-Zero Plus must be ordered as a separate standalone kit.

For any feedback or questions regarding this article (Illumina Knowledge Article #2329), contact Illumina Technical Support [e-mail].