> 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/illumina-single-cell/library-preparation-illumina-single-cell-reference_material-list/000009449.md).
# Illumina Single Cell 3' RNA Preparation library sequencing read length and loading concentrations
Illumina Single Cell 3’ RNA preparation libraries are composed of standard Illumina paired-end constructs that begin with P5 and end with P7.
Read 1 contains barcode information and must be sequenced > 45 bases.
Read 2 contains gene expression information and must be sequenced > 72 bases.
The libraries are dual-indexed with 10-base i5 and i7 indexes.
Together this totals a minimum of 137 cycles needed to sequence Illumina Single Cell 3' RNA libraries.
**Final Loading Recommendations**
The final library loading concentrations listed below are general recommendations for sequencing Illumina Single Cell 3' RNA Prep libraries. The following final library loading concentrations may need to be adjusted to optimize performance.
* NextSeq 500/550 recommended final library loading concentration 1.6 pM including ≥ 1% PhiX.
* NextSeq 1000/2000 recommended final library loading concentration 550 pM including ≥ 1% PhiX.
* NovaSeq 6000 final library loading concentration 200 pM including ≥ 1% PhiX (equivalent to Pooled Loading Concentration of 1.05 nM).
* NovaSeq X Series final library loading concentration 200 pM including ≥ 2% PhiX.
It is recommended to pool libraries together from all experimental conditions before single cell sequencing with an Illumina sequencing system as this will minimize batch effects and can help with index color balancing.
Refer to the [Supplemental Enrichment and Amplification (SEA) kit user guides](https://support.illumina.com/sequencing/sequencing_kits/illumina-single-cell-prep/documentation.html) for pooling recommendations for the applications that generate a targeted library to be sequenced with Illumina Single Cell 3' RNA libraries.
A minimum of 1% PhiX is required in the final library loading pool. If a NovaSeq X Series instrument is being used, a minimum of 2% PhiX should be used.
For more information, refer to the Sequencing section of the [Illumina Single Cell Training Packet](https://support.illumina.com/sequencing/sequencing_kits/illumina-single-cell-prep/training.html).
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9449), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009449%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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