Fig. 1 Working with individual cells a) single-cell encapsulation workflow b) reverse transcription and enrichment of full-length cDNAs c) bioinformatics pipeline d) spatial transcriptomics workflow
Differences in the transcriptomic behaviour of individual cells are not visible when heterogeneous cell populations are analysed in bulk. If the contents of a cell are labelled before analysis in such a way that molecules from each cell are distinct from one another then it is possible to compare the gene-expression level of single cells. One way of achieving this is to use water-in-oil droplets. Single cells are encapsulated in separate droplets along with a bead that is coated with reverse transcription (RT) primers. The primers surrounding any one bead contain the same cell-barcode sequence, and cell lysis and the RT reaction take place within the droplets. The result is that all cDNAs derived from the same cell share a cell barcode, and cDNAs from different cells have different barcodes (Fig. 1a). Following RT and strand switching, all cDNAs can be pooled and amplified, before attachment of sequencing adapters (Fig. 1b). During data analysis, cell barcodes are first identified by a shortlisting and error-correction process, and this label is retained throughout subsequent analyses (Fig. 1c). A similar labelling concept is used for spatial transcriptomics. Here the aim is to interpret the transcriptional activity of cells from a tissue slice in the context of their 2D location within the slice. A tissue slice is positioned over an array of RT primers, where oligos within each spot on the array have a different cell barcode. After cell permeabilisation, RT and strand-switching takes place in situ. The expression level of genes can be compared to the histological features of the slice (Fig. 1d)