Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing >50,000 single-cell genomes from diverse cell populations.
Total time
~3–5 days (cell encapsulation through sequencing library preparation and analysis)
Steps
1
Encapsulate cells in agarose microgels
Prepare and load cell suspensions into microfluidic devices to form water-in-oil emulsions, encapsulating individual cells within agarose microgel compartments for isolation and barcoding.
▶ 00:48
2
Break agarose microgels to release cells
Dissolve or disrupt the agarose matrix to liberate encapsulated single cells while preserving cellular integrity and allowing access for downstream processing.
▶ 02:48
3
Generate barcode droplets by digital PCR
Amplify and partition unique DNA barcodes into discrete droplets via digital PCR, enabling single-cell identification during multiplexed sequencing.
▶ 03:54
4
Perform tagmentation of genomic DNA in droplets
Conduct in-droplet tagmentation (enzymatic fragmentation and adapter ligation) of single-cell genomic DNA to generate sequencing-ready DNA fragments.
▶ 04:53
5
Execute single-cell barcoding by microfluidic double merger
Merge barcode-containing droplets with tagmented genomic DNA droplets using microfluidic fusion to attach unique molecular identifiers to each single-cell genome.
▶ 05:48
6
Analyze ultrahigh-throughput sequencing results
Sequence pooled barcoded libraries and computationally deconvolute reads to resolve genomes from >50,000 single cells and assess population-level heterogeneity.
▶ 08:25