rRNA Depletion using Illumina Ribo Zero Plus

The Illumina Ribo-Zero Plus rRNA Depletion 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 with the TruSeq Stranded Total RNA workflow or the Illumina Stranded Total RNA library prep kits. 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 and is compatible with 1-1000 ng of total RNA input. Public data sets for Illumina Ribo-Zero Plus can be accessed through BaseSpace.

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 Differences between the Illumina Stranded and TruSeq Stranded RNA library prep kits bulletin.

For any feedback or questions regarding this article (Illumina Knowledge Article #5899), contact Illumina Technical Support [e-mail].