Question: Can I pool Fixed RNA Profiling gene expression libraries with other 10x Genomics libraries for sequencing?
Answer: Fixed RNA Profiling gene expression libraries are dual-indexed and can be pooled with other 10x dual indexed libraries as long as the recommended sequencing configurations are followed. We have tested the pooling of Fixed RNA Profiling libraries with Single Cell 3’ v3.1 gene expression dual index libraries and saw no impact on sequencing quality when limiting Fixed RNA Profiling libraries to <40% of the total sequencing pool. It would, therefore, be low risk to pool Fixed RNA Profiling libraries with other dual index 10x libraries that have the same sequencing configuration as dual index 3' v3.1 libraries (e.g. Visium for fresh-frozen).
The following libraries can be pooled together for Illumina sequencing:
- Chromium Single Cell 3’ v3.1 (Dual Index)
- Gene Expression Libraries
- Cell Multiplexing Libraries
- Cell Surface Protein Libraries
- CRISPR Screening Libraries
- Chromium Single Cell 5’ v2 (Dual Index)*
- Gene Expression Libraries*
- V(D)J Enriched Libraries*
- Cell Surface Protein Libraries*
- CRISPR Screening Libraries*
- Visium Spatial Gene Expression Libraries (Dual Index)
- Chromium Single Cell Multiome Gene Expression Libraries (Dual Index)
*If run with 28 cycles for Read 1
The following libraries cannot be pooled for Illumina sequencing:
- Single Cell Multiome ATAC libraries
- Single Cell ATAC libraries
- Spatial Gene Expression for FFPE Libraries (Dual Index)
For additional information regarding sequencing requirements for Fixed RNA Libraries Gene Expression or Feature Barcode Libraries, please see here.
Please note: 1% PhiX is recommended for Singleplexed libraries, 5% for Multiplexed libraries, and 10% for Multiplexed libraries sequenced on NovaSeq
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex