Increase the resolution of your research
At Single Cell Discoveries, we offer a one-stop-shop service for single-cell RNA sequencing. SORT-seq (Muraro et al. 2016) is a partially robotised version of the CEL-seq2 protocol (Hashimshony et al. 2016). The SORT-seq protocol is a plate-based method, which means that single cells are sorted into prepared 384-well plates, after which the plates are processed and sequenced. For more information, please find an overview of the workflow below.
Step 1: Preparation of cell-capture plates
For single-cell sequencing based on the SORT-seq technology, we use prepared 384-well cell capture plates. The plates contain mineral oil and a tiny droplet of poly-A primers, which bind to the cell’s RNA. Additionally, the primers contain a cell-specific barcode and a unique molecular identifier (UMI), which allow us to distinguish the well-specific (and cell-specific) mRNA molecules.
The prepared cell capture plates can be ordered separately from the plate processing. When you are ready to get started, you just take the plates you need out of the freezer to start the sorting. Eventually, when you have collected all the material you need, you send the plates back to us for further processing.
Step 2: Tissue dissociation & FACS-sorting
You (our customer) dissociate the tissue of interest and sort single cells into the cell capture plates in your own facility. This means that you use your own expertise on handling the tissue of interest, as well as FACS-sort with the settings you are used to. After all; you are the expert on your tissue, while we are experts on single-cell sequencing. If required, you can also use equipment other than a FACS-machine to deposit single cells into the cell capture plates.
Step 3: Processing of the mRNA material
Once we receive the cell capture plates containing your cells, we process the plates. First, we lyze the cells, after which the barcoded primers have access to the mRNA. As a result, the poly-T stretch of the primers hybridizes to the poly-A tail of the mRNA molecules, creating RNA-DNA hybrids. Secondly, these RNA-DNA hybrids are then converted to cDNA molecules which contain the sequence of the mRNA and the primer. Because each well has a unique barcode, we can eventually pool the material from one plate into a single tube. Thirdly, the cDNA is amplified by In Vitro Transcription (IVT). Finally, we process the amplified RNA into a library for Next Generation Sequencing.
Step 4: Data analysis & -visualisation
Once the single cell transcriptome library is sequenced, we map the data to the relevant reference transcriptome. The resulting data matrices are clustered for further in-depth analysis. Various visualizations can be made with the data. First of all, a heatmap is used to show differences in gene expression for different groups of cells, whilst secondly, a t-SNE map is a way to visualize the cells and their assigned cluster number. On top of the tSNE-map, genes of interest are plotted to make sense of the data. This allows for the recognition of different cell types, rare cells or subpopulations of cells.