How does single-cell sequencing work?

There are five main steps in single cell sequencing. 

1. Produce a single-cell suspension - Tissues are dissociated into individual cells using enzymatic digestion or mechanical force. 
2. Isolate the cells - Cells are isolated individually, either through sorting with a flow cytometer or using microfluidics. Flow cytometers sort single, live cells into microwell plates, while microfluidics separate cells using droplet-based approaches. 
3. Cell marking and amplification - The small amount of mRNA in each cell is amplified using in vitro transcription (IVT) or PCR. Barcodes are added during amplification to distinguish individual cells. Following amplification, all the cDNA molecules from an individual cell will have identical barcodes. 
4. Preparing the NGS library and sequencing - The amplified and barcoded material from all cells is collected to create a NGS library. Additional sample-specific barcodes are added, and the library is sequenced. 
5. Data analysis - Raw sequencing data is compared to a reference transcriptome. Bioinformatics skills and specialized pipelines are needed for data analysis. The data is demultiplexed to the cellular level to create a count table. Single-cell analysis tools (e.g. Seurat) cluster the data based on cell-to-cell similarities, identifying cell types and subpopulations. Differential gene expression analysis reveals genes under or over-represented in clusters.