Single Cell Encapsulation for
High Throughput Sequencing: Drop Seq
Introduction to single cell encapsulation
DNA and its expression are at the heart of cellular mechanisms nevertheless our knowledge of cells and their diversity remains limited and incomplete. Its study is essential to understand the development, functioning and reproduction of living beings. Especially since a dysfunction of this point of view can be at the origin of numerous pathologies (cancer, autoimmune disease, diabetes …). Currently, the methods developed to determine and sequence the genome do not allow us to fully understand the functioning of DNA.
Evan Micosko and his team have developed a new technique to tackle this issue: The DropSeq, a single cell encapsulation for high throughput sequencing.
Principle and Process of single cell encapsulation for high throughput sequencing
Drop Seq is a method that uses microfluidics and provides the ability to target thousands of individual cells simultaneously by encapsulating them in tiny droplets. The high droplet generation frequency and the low volume that microfluidics brings allows to increase drastically the number of amplification possible by reducing the cost of consumption.
The DropSeq system uses microfluidics to generate, at a high frequency, droplets which will be used as microreactors.
In each droplet, single cell DNA and magnetic functionalized beads called “barcodes” with lysis buffer are encapsulated. Barcodes are simple DNA primers that are used to label during RT, being used as the content of the DNAs formed. Following the encapsulation of cells and barcodes, thanks to the lysis buffer, the DNA from the cell is expressed and then transcribed into mRNA. These mRNAs perform RT (Reverse Transcription) and thus mRNA copies are created as DNA.
After the RT, each droplet is broken and sequenced. The reading is done by computer software. Several DNA sequences that are copies of mRNAs of the same cell are then grouped because they have the same barcode. The RNA-Seq system allows one to know the genes that each cell individually expresses knowing that they all have the same DNA.
Protocol of droplet-based microfluidics
1. How to prepare cell suspension solutions?
To create the single cell suspension solution, a complex tissue is treated with trypsin and then dissociated into a single cell suspension.
2. Microparticles preparation and primer synthetization in microfluidic applications
Microparticles (beads) are used to deliver large numbers of distinctly barcoded primer molecules.
The primer contains:
- A constant PCR sequence to allow RNA amplification. The PCR sequence is a constant and identical sequence on all primers and beads.
- Cell barcode. Each bead contains multiple cell barcodes which are identical for all primer within the same beads but different from other primer beads. Cell barcode allows to recover the cell origin
- Unique molecular identifiers UMI, which is different on each primer within the same bead, to identify PCR duplicates
- An oligo-dT sequence for capturing polyadenylated mRNAs and priming reverse transcription
3. Barcode synthesis
A “split-and-tool” DNA synthesis strategy has been used to create massive numbers of beads with distinct barcodes. Four equal groups of microparticles and a different DNA base are added on each group: one group got the A base, another the T base etc. Then the microparticles are mixed and re-divided in 4 equal groups and a different DNA base is added on each group again. They have made this step 12 times so the primers on any given microparticles possess the same one of 412 = 16,777,216 possible 12-bp barcodes. The entire pool then undergoes eight rounds of degenerate oligonucleotide synthesis to generate the UMI on each oligo; finally, an oligo-dT sequence (T30) is synthesized on the 3’ end of all oligos on all beads.
A test has been made whose results suggested that the microparticles-of-origin for most cDNAs can be recognized by sequencing. Moreover, the results highlighted that bead contained more than 108 barcoded primer site and that the sequence complexity of the barcodes approached theoretical limits.
4. Microfluidic chip design for encapsulation
After the preparation, the cells suspension solution and beads with buffer lysis solution are injected into a microfluidic chip to be encapsulated into picoliter droplet.
The microfluidic chip is designed with two flow focusing. The first one is used to inject the cell suspension and the barcodes. Laminar flow prevents mixing of the two aqueous inputs prior to droplet formation.
The second one will generate droplet with cells and barcodes encapsulated. This chip has been designed to limit the number of double encapsulations which means to different RNA encapsulated in the same droplet. Using microfluidic allows to generate at a high frequency droplets containing beads and cell.
5. RNA hybridation process
Following the beads and the cells encapsulation the lysis buffer will break the cells and allows RNA recovering, to finally allow the hybridation to the primer.
6. STAMPs (Single-cell Transcriptomes Attached to MicroParticles)
At this step, the Rna is hybridate into the primer into the droplet. To allows cell sequencing, droplets are broken by using a specific reagent and the microparticles, which contains RNAms are collected. The RNAm is then turn into cDNA by using reverse transcription. All cDNA are recovered and called “single cell transcriptomes attached to microparticle.
7. DNA amplification using Polymerase Chain Reaction
Then the STAMPS are amplified using standard PCR in order have better signalization for high throughput mRNA-sequencing.
8. Next generation sequencing
Millions of paired-end reads are generated from a Drop-Seq library on a high-throughput sequencer. The reads are first aligned to a reference genome to identify the gene-of-origin of the cDNA. Next, reads are organized by their cell barcodes, and individual UMIs are counted for each gene in each cell. The result, shown at far right, is a “digital expression matrix” in which each column corresponds to a cell, each row corresponds to a gene, and each entry is the integer number of transcripts detected from that gene, in that cell.
Macosko et Al. calculated a capture rate of single cell into droplet of 12.8% for Drop-Seq.
He concluded by the fact that “A scientist employing Drop-Seq can prepare 10,000 single-cell libraries for sequencing in twelve hours, for about 6.5 cents per cell.”
However, he specified that all single cells isolation’s system, including Drop-Seq, is vulnerable to impurities. Even if Macosko et Al. had a biologically validated cell classification for retina analysis (100 cells per µL, allowing the processing of 10,000 cells per hour at ~10% doublet and impurity rates), he advices to use Drop-Seq in purer modes to study other tissues or applications.