# 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 that are compatible with Illumina sequencers. 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 non-coding RNA with polyadenylation). 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 reference guide](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 are partial-adapter sequences. Once they are ligated, they serve as tags for adding the index adapter sequences by PCR.

**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 captured.

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

**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#000003152%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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