> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/rna-library-prep/library-preparation-rna-library-prep-faq-list/000002329.md).

# 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 in [BaseSpace Demo Data](https://basespace.illumina.com/datacentral).

**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. Ribo-Zero Plus uses a precise targeting mechanism, and because of this, Ribo-Zero Plus may sometimes be less tolerant to bacterial species that are 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.

**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 RNA. If using Ribo-Zero Plus standalone or with the Illumina Stranded Total RNA library preparation kit, follow the [Illumina Stranded Total RNA Prep with Ribo-Zero Plus reference guide](https://support.illumina.com/downloads/illumina-stranded-total-rna-reference-1000000124514.html).

If using standalone Ribo-Zero Plus with TruSeq Stranded Total RNA library preparation, follow the reference guide for [TruSeq Stranded Total RNA with Ribo-Zero Plus](https://support.illumina.com/downloads/truseq-stranded-total-rna-with-ribo-zero-plus-rrna-depletion-ref-guide.html). Note that when used with TruSeq Stranded Total RNA, Ribo-Zero Plus must be ordered as a separate standalone kit.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2329), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002329%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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