Cost-effective single-cell transcriptomics
SORT-seq is a single-cell sequencing platform using 384-well cell-capture plates and flow cytometry (FACS) sorting.
The technology was first published in 2016 by Muraro,et al., and has since been used in numerous publications.
SORT-seq uses FACS to sort single-cells into the wells of a 384-well cell-capture plate. Each well in the plate contains barcoded primers and other reagents.
This sorting step makes it possible to analyze live single-cells, or any other particular population of interest. SORT-seq is a partially robotized version of the CEL-seq2 protocol.
Briefly, this is how it works: We receive your single cells, sorted into the wells of a prepared 384-well cell capture plate.
Then, RNA molecules are barcoded with primers that contain a cell-specific barcode.
After barcoding, double-stranded cDNA is generated, then we pool the contents of the wells and start the amplification of the RNA with in-vitro transcription (IVT).
Once IVT is complete, we prepare the sample into an Illumina sequencing library.
What you can do with SORT-seq
The SORT-seq platform is a suitable solution for biological questions that don’t require tens of thousands of cells per sample.
Furthermore, the flexible plate-based system makes it possible to screen small numbers of cells from many different samples, without skyrocketing costs.
Finally, you can combine single-cell flow cytometry data with transcriptomics data with strategies like GateID to find novel FACS gating strategies for desired cell types.
Key benefits of SORT-seq
- Cost-effective single-cell sequencing
With prices starting at just over €1000 per sample, SORT-seq is one of the most cost-effective single-cell sequencing methods available.
- Modular and flexible due to the use of plates
Working with plates allows you to start small and upscale later. We can start by processing and sequencing one plate per condition. Once the data from the first plate looks good, we can go on to process the rest of the plates. This provides you with tremendous flexibility in scaling up your experiments.
- Compatible with all sample types
SORT-seq works even with rare or low quantities of cells. As long as you can fill at least part of a plate with FACS sorting, you can generate single-cell sequencing libraries. Also, SORT-seq can handle cells of all sizes. Cells that are too big for the 10x Genomics system (e.g. cardiomyocytes) can be analyzed with SORT-seq without any issues.
Step 1: FACS sorting
Begin by ordering our cell capture plates, which we ship worldwide. You then sort your single-cells into the 384-well cell-capture plates and send the plates back to us for processing. If you don't want us to process all the plates at once, just let us know which ones to start with.
Step 2: Plate processing
Once we've received your plates, we'll process them using the SORT-seq protocol. After barcoding the mRNA of your single-cells, we'll pool the material from one plate to generate one sequencing library per plate.
Step 3: Sequencing and data analysis
We'll send the sequencing libraries to one of our partners for sequencing. When we get the data back, we'll perform a preliminary data analysis, then send the data back to you.
As with all of our services, we provide ongoing support from our single-cell sequencing specialists. For each SORT-seq project, we provide the following:
- Regular email updates during plate processing
- Raw sequencing data (FASTQ files)
- A QC report per plate
- Mapped count tables for downstream analysis
- A preliminary clustering analysis, done with the Seurat pipeline.
- A preliminary differential gene expression analysis
Once we have sent you the data, we'll invite you to schedule a call with one of our scientists to go over any additional questions you may have.
That's when we can discuss any additional data analysis you may need.
We'll need a minimum of two plates for each sample or condition. This ensures there is always a technical back-up plate available in case something goes wrong with the shipment or processing.
This mostly depends on your biological question. For cell type identification in a sample that contains only a few cell types, 2-3 plates are probably enough. If you are interested in subtle changes in the transcriptome, or if you are looking for rare cell populations, we may need more plates.
Yes, you can first process one plate (or set of plates) and then continue with the rest later on. Any minor technical batch effects should be negligible, especially if the biological differences between populations of cells in your sample are clear.
The first step is to reach out to us to set up a meeting with one of our specialists. During this meeting we'll discuss your experiment and help you define a pilot experiment. Next, you order the cell capture plates via our website. Once you have sorted single-cells into the plates, you ship them back to us for processing.
While this is possible in principle, it will likely require some batch effect correction. Different technologies will yield slightly different results due to technology-specific technical quirks and other biases. However, with batch effect correction and a clear biological difference between the different populations in your samples, it should be possible to mix with data from other platforms.
Not sure if SORT-seq is right for your project? Explore our other services.
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