- Things to consider before starting with single-cells:
- 10X Genomics: 3’mRNA-Seq
- Fluidigm C1:
- Nano-litre dispensing:
We have two single-cell systems from 10X Genomics and Fluidigm. These can both be used to perform single-cell RNA-Seq which is the application most users are interested in. We are currently working on making these services that are as robust and easy to use as our standard RNA-seq or exome pipelines.
Please do 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:
- You need single cells – obvious but important. Make sure you don’t have cell clumps, and tissue disaggregation must be done very carefully.
- Count your cells – cell concentration is important, particularly in droplet platforms. Use a hemocytometer, or the Countess in Flow Cytometry.
- 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 that mRNAs can be converted to cDNA and tagged with a barcode that informs the user which cell the RNA-Seq reads came from. Around 1,000 to 10,000 cells are captured in just 10 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, so nearly 10% if you go for the maximum 10,000 cells per sample.
Costs: £1500ish per sample* £1200 per sample for the single-cell 3’mRNA-Seq library prep. £200 for 1000 cells at 50,000 reads each (assuming sample multiplexing for NovaSeq sequencing).
Based on Fluidigm microfluidic technology, the C1 Single-Cell Auto Prep System enables rapid isolation, processing and profiling of individual cells for genomic analysis. The single-cell gene expression workflow allows processing of 96 single cells, which are captured, lysed, verified, reverse transcribed and preamplified. At the end of this process two workflows can be followed: direct gene expression of full-length cDNA through qPCR using the Biomark, or library preparation using Nextera XT (for this you will need Clontech SMART Ultra Low for Fluidigm C1.) The machines are bookable on resource Scheduler (Josephine and Daphne. You will have to ensure that you have booked the bioanalyser if you plan to verify cDNA amplification from each cell, please give yourself enough time to run up to 96 samples. Lastly you will also need to contact Microscopy to discuss the use of an appropriate microscope to view single-cell capture sites in the C1. We can offer a library prep service for C1 templates using Nextera XT; if you are interested please get in touch.
Doublet rate: around 1-5% depending on chip type used.
Costs: £1200ish per sample* £500 per Fluidigm chip plus £500 for reagents. £200 for 1000 cells at 50,000 reads each (assuming multiplexing samples in a HiSeq 4000 lane)
Instruments are available to book on Resource Scheduler but you will need training and we’d recommend a design meeting too. We are looking into offering a full service on the C1 – watch this space.
Hacking the C1: The Fluidigm C1 can be modified to run chemistry or applications you choose. We’ve purchased licences to do this so please come and talk to us about what’s possible. The ScriptHub website allows you to see what other C1 users around the world are trying to do, upload your protocols to share them.
Currently methods for ATAC-seq, CAGE, mRNA-seq, STRT-seq and CEL-seq are available.
Drop-seq is a technology that allows biologists to analyse genome-wide gene expression in thousands of individual cells in a single experiment. This work is described in Macosko et al., Cell, 2015. The technology is being used on another lab in Cambridge and we are looking into whether the Drop-seq technology can be brought into the Genomics Core.
We have previously shown that cells can be flow-sorted and sequenced using standard library-prep kits. We are looking at bringing in nanolitre liquid handling technology to the Genomics Core such as the LabCyte Echo and/or Mantis systems.
*sorry about the “ish” next to costings but single-cell experiments are so variable we can only really tell you how much your experiment might cost once we understand the details.