What concentration should I load my libraries at for sequencing?

Question: What concentration should I load my libraries at for sequencing?

Answer: Our recommendations for the optimal loading concentration, as well as pooling considerations, are listed in the sequencing section of each assay User Guide. Please ensure that the correct guide is used for the assay. Examples are shared below:

Single Cell Gene Expression (v3.1)

Single Cell Immune Profiling (5’ v2)

Single Cell ATAC and Single Cell Multiome ATAC & Gene Expression

Fixed RNA Profiling

Spatial Gene Expression (Fresh Frozen and FFPE)

Targeted

Library concentration (nM or pM) should be determined by Kapa Library quantification kit (part no. KK4824) and size corrected using fragment analysis. For further details, please see: Why do I need to use qPCR to quantify my final library?

While these loading concentrations have worked well in-house, there may be some optimization required for inherent differences between sequencers. We recommend working with Illumina Technical Support to optimize cluster density. 

Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC+GEX, Single Cell ATAC, Fixed RNA Profiling, Visium for FFPE, Visium for FF, Visium CytAssist for FFPE, Targeted