Question: How can I generate enough library mass to use as input into the Targeted Gene Expression assay?
Answer: For the Targeted Gene Expression assay, we recommend using approximately 300ng of each Whole Transcriptome Analysis (WTA) library as input.
If insufficient WTA library material is available, the library can be amplified using the 10x Library Amplification kit. This protocol starts with 20 ng of WTA library and typically yields ~1-2 ug of re-amplified WTA library. Please refer to the protocol provided in the Appendix of the Targeted Gene Expression User Guide.
We have found that library re-amplification leads to a slight reduction in library complexity (2-4% loss of UMIs), but cell calling, cell clustering and library quality are minimally or not affected.
Adding a cycle during SI PCR
As an alternative strategy to generate additional WTA library mass, one extra PCR cycle can be added to the Sample Index PCR step during WTA library preparation.
We have tested this approach on Single Cell 3' v3.1 Dual Index Gene Expression libraries and Visium Spatial Gene Expression libraries and found that adding one extra cycle to the SI PCR had no impact or only minimally impacted library complexity, library quality, and clustering. This approach has not been tested with Single Cell 5' Gene Expression libraries.
We do not recommend adding more than one extra PCR cycle at this step.
Products: Targeted Gene Expression, Single Cell Gene Expression, Single Cell Immune Profiling, Visium