We provide expertise in single-cell experimental design, library preparation, sequencing and bioinformatics analysis. There are multiple different methods that can be used for single-cell transcriptomics. At the Genomics Core Leuven we offer three complimentary techniques:
- 10x genomics chromium Single Cell 3′
SMARTseq offers the highest sensitivity through capture of full length poly-A RNAs.
- Most sensitive
- Full length cDNA allows splicing and SNP detection
- Can be combined with FACS sorting for direct isolation of rare cells
- Can be combined with FACS for index sorting
- High yield of cells (75 – 90%) from a single-cell suspension using FACS.
- High cost per cell
- Low throughput (100s of cells)
- High sequencing requirements (optional)
10x genomics chromium Single Cell 3′ is a commercial solution to droplet based 3’ tag sequencing
- High throughput (1000s of cells)
- Up to 8 different samples on 1 chip
- Medium cost per cell
- Medium yield of cells from a single-cell suspension (up to 65%)
- Low sequencing requirements
- Medium sensitivity
- Only 3’ end of cDNA sequenced
Dropseq is a custom built microfluidics device for droplet based 3’ tag sequencing
- Lowest cost
- High throughput
- Customizable chip design
- Low efficiency of cell capture (10%)?
- Lowest sensitivity
- Requires custom sequencing primers
Which technique to use?
The technique to use depends on the aim of the experiment. For deep characterization of a limited number of cells into SMART-seq is an ideal technology. Due to the high sensitivity of the technique genes with lower expression (e.g. transcription factors) can be detected and used to find subtle changes in phenotype.
For tissue based profiling where the goal is to identify distinct cell types in a complex mixture a droplet technology is best. Due to the high throughput it is possible to identify rare cell types in a subpopulation. Due to the low sensitivity only highly expressed genes and major gene expression programs are detected. Highly expressed genes may be used to identify markers for subsequent characterization.
A consultation with the single-cell genomics core is advised before deciding on an experimental approach.