Question: Is the performance of the 5' Gene Expression Solution Equivalent to 3' v2 for Transcriptome analysis?
Answer: The 5' and 3' gene expression kits capture different information from the transcript, although both amplify full-length cDNA using a polyd(T) and switch oligo primers. In the Single Cell 3' assay, the polyd(T) oligo sequence is on the gel bead and the switch oligo is provided in the Master Mix, while in the Single Cell 5' gene expression assay, the switch oligo is on the gel bead and the polyd(T) is provided in the Master Mix. Please refer to the article "#What is the difference between a Single Cell 3' and 5’ Gene Expression library?" for more information.
The 3' and 5' assays provide comparable sensitivity in terms of genes and UMIs captured per cell. We don't have a direct comparison between 3' and 5' gene expression data currently available, although we have 3' and 5' gene expression datasets for PBMCs available on the 10x website that you may be interested in exploring:
The Single Cell 3' solution is recommended for general gene expression applications, as 3' is the gold standard and the better-understood approach in the scientific community. For this reason, the human transcriptome reference may be more complete at the 3' end. If you plan on profiling your samples based on VDJ transcripts as well, then the 5' solution is recommended.
Although the data generated is comparable, the choice of solution is also dependent on your research question.
- The 5’ assay generates reads at or close to the transcription start site of each gene, and informs TSS location and potentially alternative promoter usage.
- The 3’ assay generates sequence close to gene transcription termination and polyadenylation sites, which may reveal 3’ UTRs, their genomic location and alternative terminal sequences. In addition, the 3’ assay may facilitate studies into post-transcriptional regulatory mechanisms e.g. 3’ UTR miRNA binding sites
Products: Single Cell Gene Expression, Single Cell Immune Profiling