> 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/000003290.md).

# Illumina Stranded mRNA Prep Ligation Protocol FAQs

**Can any thermal cycler be used?**

Illumina recommends one of the following thermal cyclers:

* Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad, catalog # 1851196)
* T100 Thermal Cycler (Bio-Rad, catalog # 1861096EDU)

Other thermal cyclers have not been validated by Illumina, but may be compatible; users must validate alternative thermal cyclers in their own labs. Alternative thermal cyclers must have a heated lid temperature setting as some steps require the lid to be set at 40ºC. Ensure that the thermal cycler meets this requirement.

**Are there any intermediate QC steps?**

No, there are no intermediate QC steps.

**How is strandedness retained?**

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)

**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?**

Libraries can be checked using the Agilent 2100 Bioanalyzer and DNA 1000 Kit or the TapeStation System and D1000 ScreenTape.

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

Illumina has validated storing dsDNA libraries at -20ºC for up to 30 days. It is recommended to perform library QC and quantitation immediately prior to sequencing.

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

The average fragment length is \~300-400 bp. The expected insert size is \~160 bp.

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

This could indicate the presence of bubble products; 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.

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                              |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #3290), 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)*.* |


---

# Agent Instructions
This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com.

## Querying This Documentation
If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter:

```
GET https://knowledge.illumina.com/library-preparation/rna-library-prep/library-preparation-rna-library-prep-faq-list/000003290.md?ask=<question>&goal=<endgoal>
```

`ask` is the immediate question: it should be specific, self-contained, and written in natural language.
`goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal.

The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.