Drop-Seq

Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015 May 21;161(5):1202-14. doi: 10.1016/j.cell.2015.05.002.

Contact: abasu@broadinstitute.org

Cells, the basic units of biological structure and function, vary broadly in type and state. Singlecell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single cell resolution.

Molecular barcoding of cellular transcriptomes in droplets (A) Drop-Seq barcoding schematic. A complex tissue is dissociated into individual cells, which are then encapsulated in droplets together with microparticles (gray circles) that deliver barcoded primers. Each cell is lysed within a droplet; its mRNAs bind to the primers on its companion microparticle. The mRNAs are reverse-transcribed into cDNAs, generating a set of beads called “single-cell transcriptomes attached to microparticles” (STAMPs). The barcoded STAMPs can then be amplified in pools for high-throughput mRNA-seq to analyze any desired number of individual cells. (B) Sequence of primers on the microparticle. The primers on all beads contain a common sequence (“PCR handle”) to enable PCR amplification after STAMP formation. Each microparticle contains more than 10⁸ individual primers that share the same “cell barcode” (panel C) but have different unique molecular identifiers (UMIs), enabling mRNA transcripts to be digitally counted (panel D). A 30 bp oligo dT sequence is present at the end of all primer sequences for capture of mRNAs. (C) Split-and-pool synthesis of the cell barcode. To generate the cell barcode, the pool of microparticles is repeatedly split into four equally sized oligonucleotide synthesis reactions, to which one of the four DNA bases is added, and then pooled together after each cycle, in a total of 12 split-pool cycles. The barcode synthesized on any individual bead reflects that bead’s unique path through the series of synthesis reactions. The result is a pool of microparticles, each possessing one of 4¹² (16,777,216) possible sequences on its entire complement of primers. (D) Synthesis of a unique molecular identifier (UMI). Following the completion of the “split-and-pool” synthesis cycles, all microparticles are together subjected to eight rounds of degenerate synthesis with all four DNA bases available during each cycle, such that each individual primer receives one of 4⁸ (65,536) possible sequences (UMIs).