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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.

Dolomite_Bio_scRNAseq workflow_ Drop-Seq

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.

scRNAseq workflow 2 Drop-Seq

  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 tick
Flexible buffer composition tick tick
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
tick tick
Possibility to run samples
in parallel
tick(up to 8) X
Sample cooling tick X
Gentle agitation of cells and beads tick 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.

Learn more about scRNA-Seq on NadiaCTA Find out more - Drop-Seq