> 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/000003291.md).

# Sequencing FAQs for Illumina Stranded RNA libraries

The information in this article pertains specifically to the Illumina Stranded mRNA and the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (or Ribo-Zero Plus Microbiome) library prep kits.

**What are the advantages of performing paired-end sequencing of RNA libraries?**

Paired-end sequencing can help with data alignment, discovery of novel transcripts, and identification of PCR duplicates.

**What are the suggested loading concentrations for the various sequencers?**

Recommended loading concentrations can be found in either the [Illumina Stranded mRNA Reference Guide](https://support.illumina.com/downloads/illumina-stranded-mrna-reference-1000000124518.html), [Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Reference Guide](https://support.illumina.com/downloads/illumina-stranded-total-rna-reference-1000000124514.html), or [Denature and Dilute Protocol Generator](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm) workflow for each instrument. Loading concentrations may require optimization within each lab.

Note that Illumina Stranded RNA libraries require dark cycles recipes or sufficient PhiX spike-in during sequencing. See the following resources for more information:

* [Custom recipes for Illumina Stranded libraries on NextSeq 1000/2000](/instrumentation/nextseq-1000-2000/instrumentation-nextseq-1000-2000-faq-list/000005583.md)
* [Custom recipes for Illumina Stranded Total RNA and mRNA libraries on NovaSeq X Series](/instrumentation/novaseq-x-x-plus/instrumentation-novaseq-x-x-plus-reference_material-list/000008394.md)
* [PhiX requirements for sequencing Illumina Stranded RNA libraries](/library-preparation/rna-library-prep/library-preparation-rna-library-prep-reference_material-list/000003708.md)

**Which strand is sequenced in Read 1?**

Read 1 sequences will be identical to the antisense strand of DNA, which is also the first strand cDNA.

**Can libraries prepared with this kit be combined with other library types in the same flow cell lane?**

This is not supported.

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                              |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #3291), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003708%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/000003291.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.