> 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/000003152.md).

# Illumina Stranded Total RNA Prep, Ligation with Ribo Zero Plus FAQs

**What is the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit?**\
The Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit generates stranded total RNA libraries. The workflow is based on rRNA depletion and targets long RNA species (primarily mRNA and long non-coding RNA) for library preparation. Small RNAs will not be faithfully retained.

**Which species is the kit compatible with?**\
The kit is compatible with human, mouse, rat, and bacterial total RNA. *E. coli* (gram-negative) and *B. subtilis* (gram-positive) have been tested. Performance may vary when using samples from other species.

**Why choose to sequence total RNA instead of mRNA?**\
The choice of the application will depend on the project's aim. mRNA selection will specifically target poly-adenylated RNA (including coding RNA and polyadenylated non-coding RNA). Total RNA kits prepare RNA libraries after rRNA depletion, targeting coding RNA and long non-coding RNA.

**Which reagents are needed to be ordered to complete the workflow?**\
The [Illumina Stranded Total Product Documentation](https://support.illumina.com/downloads/illumina-stranded-total-rna-reference-1000000124514.html) lists the required Illumina kits and user-supplied consumables that are required for this application.

**What is the purpose of the index anchors?**\
The anchors add partial adapter sequences. Once they are ligated, they serve as tags for adding the rest of the index adapter sequences via PCR. See [Understanding the differences between Illumina index chemistries and anchor function](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Understanding-the-differences-between-Illumina-index-chemistries-and-anchor-function-support-bulletin/README.md) for more details.

**Are TruSeq RNA index adapters compatible with this kit?**\
No.

**Which RNA molecules are targeted for inclusion in the final libraries with this kit?**\
This kit will mainly target mRNA and long non-coding RNAs. Small RNAs will not be faithfully retained.

**Which RNA molecules are depleted with this kit?**\
The Ribo-Zero Plus rRNA removal mix targets cytoplasmic rRNA, mitochondrial rRNA, bacterial rRNA, and human globin mRNA (suitable for blood and non-blood samples).

Ribo-Zero Plus Microbiome additionally targets 5S, 16S, 23S from diverse microbes including common adult, infant bacterial species and ATCC MSA-2002, MSA-2005, and MSA-2006 rRNA targets.

**Does the kit deplete globin mRNA from non-human total RNA?**\
The Ribo-Zero Plus rRNA removal mix has been validated to deplete globin mRNAs from human samples; other species have not been validated.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3152), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000006561%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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