How does it work
The drop-seq method was originally developed by Macosko et al. in 2015 and has since emerged to one of the most popular single cell RNA-Sequencing (scRNA-Seq) methodologies.
Core Principles of Drop-seq
- Encapsulation of single cells with barcoded mRNA-capture beads
- The use of a microfluidic device/chip to create nanoliter-scale aqueous droplets formed by precisely combining aqueous and oil flows
- The use of molecular barcoding beads to distinguish the cell-of-origin of each mRNA
- Reverse transcription added post encapsulation using a template switching oligo
The methodology involves encapsulating single cells with single barcoded mRNA-capture beads in nanoliter-sized droplets. Beads are suspended in lysis buffer that upon encapsulation with cells inside a droplet, break open the cell and thereby releases the mRNA.
The barcoded oligo bead library is constructed such that each bead has a unique DNA barcode sequence, but within a bead, the thousands of copies of oligo all contain an identical barcode sequence. The 3′ end of the oligo has a poly(dT) stretch that captures mRNA and primes reverse transcription.
Subsequently, beads are then released from their droplets and subjected to reverse transcription. The cDNA product is a full-length amplicon of the mRNA transcript due to the use of template switching oligonucleotide. Further on, the cDNA is amplified and sequenced using conventional library preparation methods and Next Generation Sequencing such as offered by Illumina or PacBio.
Droplet Based scRNA-Seq Methods
Dolomite Bio’s Nadia instrument seamlessly adopts the Drop-seq protocol. However, with the flexible and open nature of the system, there is also potential for alternative droplet-based methods to be adopted including scRNA-seq using soft beads, ppRNA-Seq, Dronc-seq, Perturb-seq and CROP-seq
The popularity of Drop-seq is owing to several benefits:
- High throughput – 5,000 – 50,000 single cell transcriptomes per run
- High capture rate of around 11% of transcriptomes of cells
- Low doublet rate of about 5%
- Commercial availability of the barcoded beads
- Relatively low cost