# Illumina Stranded mRNA Prep Ligation Sequencing FAQs

**What is the recommended read length and number of reads?**

The read length and format (single read versus paired-end) are important considerations in the design of RNA sequencing experiments. The table below provides guidance, but do not represent strict cut-offs or official recommendations. Needs for individual projects may vary based on multiple variables as well as user preference. Consult the literature for the most up to date resources. (M = million)

|                                                         |               |                                            |
| ------------------------------------------------------- | ------------- | ------------------------------------------ |
| **Application**                                         | **Read Type** | **Read Depth per sample (mRNA/Total RNA)** |
| Gene profiling (gene-level counts)                      | 1 x 51        | >5 M / >10 M                               |
| Differential gene expression                            | 2 x 76        | 30-60 M                                    |
| Discovery (alternative transcripts, gene fusions, etc.) | 2 x 76        | ≥50 M / >100 M                             |
| Complete transcriptome annotation                       | 2 x 76-101    | ≥100 M / ≥200 M                            |

**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?**

Follow the guidelines in the specific reference guides of the instrument; loading concentrations may require optimization within each lab.

**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.

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