# rRNA Depletion using Illumina Ribo Zero Plus
The [Illumina Ribo-Zero Plus rRNA Depletion](https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html) kit uses an enzymatic depletion method that targets human, mouse, and rat cytoplasmic and mitochondrial rRNA, globin transcripts, and bacterial rRNA with one reagent. Table 1 shows the RNA species targeted for depletion.
| **Sample** | **Targeted RNAs** |
| ----------------------------- | --------------------------------- |
| 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 | *Escherichia coli:* 5S, 16S, 23S |
| Gram (+) Bacterial rRNAs | *Bacillus subtilis:* 5S, 16S, 23S |
Table 1. RNA species targeted for reduction.
Ribo-Zero Plus uses DNA probes and enzymatic depletion to remove abundant transcripts from total RNA samples; whereas legacy Ribo-Zero kits use biotinylated RNA probes to pull down abundant transcripts with streptavidin beads (see Figure 1).
Figure 1. Ribo-Zero Plus and legacy Ribo-Zero depletion methods.
Ribo-Zero Plus can be used as a [standalone kit](https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html) with the [TruSeq Stranded Total RNA](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-stranded-total-rna.html) workflow or the [Illumina Stranded Total RNA library prep kits.](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/stranded-total-rna-prep.html) The Ribo-Zero Plus reagent can be purchased individually or bundled with Illumina Stranded Total RNA. Ribo-Zero Plus does not come bundled with TruSeq Stranded Total RNA kits. If using Ribo-Zero Plus with the TruSeq Stranded Total RNA kit, Ribo-Zero Plus rRNA depletion reagents need to be purchased separately. The Ribo-Zero beads that come bundled with the TruSeq Stranded Total RNA kit are not used.
| Workflow | Compatible with
Ribo-Zero Plus
| Ribo-Zero Plus
bundled with kit
| Special considerations for use |
| --------------------------- | ---------------------------------------- | ----------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| TruSeq Stranded Total RNA | Yes | No | Use TruSeq Stranded Total RNA with
Illumina Ribo-Zero Plus rRNA depletion reference guide
|
| Illumina Stranded Total RNA | Yes | Yes | None |
Table 2. Ribo-Zero Plus kit compatibility and bundling options.
When paired with the Illumina Stranded Total RNA kit, Ribo-Zero Plus offers key advantages that enable enhanced study flexibility:
* “One tube (single kit) to deplete multiple species” functionality
* Enhanced depletion at lower total RNA inputs
* A more precise mechanism for depletion of targeted transcripts
The protocol for rRNA depletion is found in the [Illumina Stranded Total RNA reference guide](https://support.illumina.com/downloads/illumina-stranded-total-rna-reference-1000000124514.html) and is compatible with 1-1000 ng of total RNA input. Public data sets for Illumina Ribo-Zero Plus can be accessed through [BaseSpace](https://basespace.illumina.com/datacentral).
Custom probes can be added to the Ribo-Zero Plus workflow to target different species. The Ribo-Zero Plus custom design process involves inputting a list of target RNA species through an internal BaseSpace App. The BaseSpace App provides additional probe designs that can be added to Ribo-Zero Plus to deplete specified targets. For more information on the Ribo-Zero Plus custom design process, contact your local Illumina sales team. Probe pools will be ordered from a third party.
For more information on differences between Illumina Stranded Total RNA and TruSeq Stranded Total RNA, refer to the [What are the main differences between TruSeq Stranded and Illumina Stranded kits?](https://knowledge.illumina.com/library-preparation/rna-library-prep/library-preparation-rna-library-prep-faq-list/000005928) article.
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