Question: How do I design a custom PCR-based target enrichment assay for Single Cell V(D)J?
Answer: While we recommend using our hybrid/capture-based Targeted Gene Expression product to enrich Single Cell 5’ Gene Expression libraries for specific genes, it may also be feasible to implement a PCR-based target enrichment approach.
Please note that custom PCR-based target enrichment approaches have not been tested or validated by 10x Genomics, and are not supported. We are unable to provide specific experimental guidelines, and can only provide the general recommendations included here.
For PCR-based target enrichment, It may be possible to design custom enrichment primers to amplify targets of interest during the Single Cell V(D)J workflow, in place of the Human or Mouse T Cell Mix 1 & 2 or Human or Mouse B Cell Mix 1 & 2 which amplify the T-cell Receptor (TCR) or Immunoglobulin (Ig) transcripts, respectively.
To develop a targeted assay, we recommend consulting the 'Introduction' and 'Appendix: Oligonucleotides' sections of the Chromium Single Cell V(D)J Reagent Kits User Guides, which details the Single Cell VDJ assay scheme, primer sequences, as well as enrichment primer concentrations, for guidance:
Note that TCR and Ig genes are typically expressed at low levels and therefore the PCR conditions and cycling may also need to be optimized depending on the expression level of the mRNA target(s). Genes with low levels of expression are not good candidates for targeted sequencing since they are unlikely to be detected at a high enough level to give adequate representation across cells in the sample.
A few additional notes:
- The number of Target Enrichment cycles may need to be increased by 1-2 cycles, particularly if there is a low relative abundance of the transcript of interest.
- The number of SI-PCR cycles will likely need to be increased by 1-2 cycles since the total initial mass will be significantly less.
- Depending on the length of the transcript(s) of interest, the parameters for fragmentation may need to be optimized to prevent excessive fragmentation. Fragmentation has been optimized for cDNA originating from full-length mammalian transcriptomes and thus fragmentation from a selected group of transcripts may require protocol adjustments. The length of the fragmentation reaction (2 minutes) can be increased or decreased accordingly.
Products: Single Cell Immune Profiling