> 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/000003286.md).

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

**Can any thermal cycler be used?**

The thermal cycler must have a heated lid temperature setting as some steps require the lid to be set at different temperature than default (for instance 40°C). Furthermore, one step requires a gradient decrease of 0.1°C/second. Verify that the thermal cycler meets these requirements.

**3** **Are there any** **intermediate QC steps?**

No, there are no intermediate QC steps recommended in the reference guide.

**4** **How is strandedness maintained in the libraries?**

See [Which reads map to each strand in Stranded RNA workflows, and how is strandedness achieved?](/library-preparation/rna-library-prep/library-preparation-rna-library-prep-reference_material-list/000002238.md) for more information.

**How to choose optimal index combinations?**

For optimal index balancing, see the [Index Adapters Pooling Guide](https://support.illumina.com/downloads/index-adapters-pooling-guide-1000000041074.html).

**What is the recommended final library QC method?**

Analyze 1 µl library using one of the following:

* Agilent 2100 Bioanalyzer and DNA 1000 Kit
* TapeStation System and D1000 ScreenTape

**How to** **store final libraries and for how long are they stable?**

Illumina has tested storing double stranded DNA libraries at -20ºC for up to 30 days. It is also recommended to perform library QC and quantitation immediately prior to sequencing, following any storage time.

**9** **What is the expected final library size?**

The average fragment length is \~375-475 bp.

**What is the shoulder of higher molecular weight that occasionally appears in library traces?**

This could be bubble product. See [Bubble products in sequencing libraries: causes, identification, and workflow recommendations](/library-preparation/general-library-prep/library-preparation-general-library-prep-reference_material-list/000001918.md) for more information.

**How long does it take to generate ready-to-sequence libraries?**

Typically, ≤8 hours total turnaround time.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3286), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001918%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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