Dropseq

Drop-seq is a high-throughput single-cell RNA sequencing method that enables expression profiling of thousands of individual cells in a single run. It uses droplet microfluidics to co-encapsulate single cells with barcoded beads, allowing each cell's transcripts to be tagged with a unique barcode for downstream sequencing.

In brief, a suspension of single cells and barcoded beads is flowed into a microfluidic device that generates nanoliter droplets. Ideally, each droplet contains one cell and one bead. Inside the droplet, cell lysis releases mRNA, which binds to barcoded oligonucleotides on the bead. Reverse transcription occurs within the droplet, producing cDNA labeled with a cell-specific barcode and a unique molecular identifier (UMI). Droplets are broken, cDNA is pooled, amplified, and sequenced.

Data processing involves aligning reads to a reference genome, collapsing reads by UMI to count transcripts per gene, and constructing a gene expression matrix with cells as columns and genes as rows. Downstream analyses include quality control, normalization, dimensionality reduction, clustering, and cell type annotation.

Applications include broad profiling of complex tissues to identify cell types and states, developmental trajectories, and rare cell populations in immunology and neuroscience. Advantages include high throughput and relatively low per-cell cost, while limitations include potential doublets, ambient RNA contamination, capture bias toward polyadenylated mRNA, reliance on specialized microfluidic equipment, and relatively shallow per-cell sequencing depth.

Drop-seq has spurred numerous adaptations and inspired later high-throughput single-cell methods that use barcoded beads and droplets, contributing to the rapid expansion of single-cell transcriptomics.