We have two single-cell workflows, from 10X Genomics, and plate based library prep. These can both be used to perform single-cell 3′ mRNA-seq for gene expression (the application most users are interested in) as well as 5′ gene expression, V(D)J targeted for immunological studies, ATAC-seq, CITE-seq, and Copy Number.

Come and talk to us about accessing these technologies, or about how your experiments may benefit from looking at single-cell resolution.

Things to consider before starting with single-cells:

It sounds almost too obvious, but the most important thing you can do to start well is develop a robust and reliable method for dissociating and counting your cells. You know how your cells behave better than anyone, and the method you choose may be specific to your cells.

• You need single cells – obvious but important. Make sure you don’t have cell clumps, and tissue dis-aggregation must be done very carefully.
• Count your cells – cell concentration is important, particularly in droplet platforms.
• How many cells do you need – this is difficult to give a simple answer to, come and talk to us.
• How deeply should you sequence – this is difficult to give a simple answer to, come and talk to us. We suggest using 50,000 reads per cell.
• How much will it cost – single-cell experiments are not cheap. Some prices are indicated below but it very much depends on how many cells and how deeply you sequence them.
• Will “doublets” be a problem – doublets are when two cells are captured in what should be a single cell experiment. Whether these will affect your analysis or not is dependant on how many doublets you have, and the sort of question you are trying to answer. Come and talk to us.

10X Genomics: 3’mRNA-Seq v3

10X Genomics captures single-cells in droplets so the mRNAs can be converted to cDNA and tagged with a barcode that informs the user which cell the mRNA-Seq reads came from. Around 1,000 to 10,000 cells are captured in just 20 minutes using the following workflow: capture cells with beads, release RNA, oligo-dT primed cDNA to get a 3′ mRNA-seq library.

Doublet rate: around 1% per 1000 cells captured, but up to 8 – 10% if you go for the maximum capture of 10,000 cells from a sample.

Costs: ~£1500 per sample* £1200 per sample for the single-cell 3’mRNA-Seq library prep. £200 for 1000 cells at 50,000 reads each (assuming samples are multiplexed in a NovaSeq lane).

*apologies for the non-exact costings, but single-cell experiments are so variable we can only really estimate how much your experiment might cost once we understand the details, typically that is the number of cells you aim to capture, and the read depth required.

Nano-litre dispensing:

We and others have shown that cells can be flow-sorted into 384 plates and sequenced following standard library-prep (at a reduced reagent volume) with low volume liquid handling, e.g., our SPT labtech Mosquito HV.