Single Cell RNA Sequencing with Drop-Seq
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, PacBio or Oxford Nanopore Technologies.
| scRNA-Seq on the Nadia Instrument |
Drop- Seq | |
| Type of microfluidic chip | Standard, highly precise Nadia chip | Custom PDMS chip |
| Probability of chip blockage | Very low | High |
| Bead type | Deformable (soft) or non-deformable (hard) |
Non-deformable |
| Capture rate | 11% with hard beads 50-70% with soft beads |
10% (hard beads) |
| Bead recovery method | Filtration (<10 mins) | Centrifugation and phase separation (>30 mins) |
| Doublet rate | Low | Variable due to lack of stirring (depending on cell type) |
| Lysis efficiency | High |  |
| Flexible buffer composition | |
|
| Total cell captures/ run | 50k | n/a |
| Median number of genes / cell* |
5.5k | 4.8k |
| Median number of transcripts / cell* |
23.2k |
16.3k |
| Possibility to sub-sample cells for sequencing |
|
|
| Possibility to run samples in parallel |
(up to 8) |
X |
| Sample cooling | |
X |
| Gentle agitation of cells and beads | |
X (for cells, stir bar for beads) |
| Total length of protocol | 9h | 12h |
*sequencing depth: 120k reads / cell
Lear more about the scRNA-Seq protocol in this step by step video tutorial
ScRNA-Seq on the Nadia Instrument
Dolomite Bio’s Nadia Instrument seamlessly adopts the Drop-seq protocol. The Nadia Instrument is an automated, microfluidic droplet-based platform for single cell research that encapsulates up to 8 samples, in parallel, in under 20 mins. Over 50,000 single cells can be captured per cartridge in a run.
