Microfluidics-free single-cell genomics with templated emulsification

Abstract

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse–human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Exceeding 80% Efficiency of Single-Bead Encapsulation in Microdroplets through Hydrogel Coating-Assisted Close-Packed Ordering

“…If needed, scaling up the vortex approach is straightforward, simply requiring a larger vortex mixer to hold multiple tubes for parallelization. Such scalability is highly desired for ultrahigh-throughput single-cell or digital diagnostic assays, 44 especially those undergoing a commercialization pipeline.…”

Slide-tags: scalable, single-nucleus barcoding for multi-modal spatial genomics

“…We estimate that the combination of dissociation and microfluidic losses during nuclei barcoding collectively account for ~75% of the nuclei lost. This represents an avenue for significant improvement through tissue-specific optimizations to the dissociation, and improved droplet microfluidics or, potentially, microfluidic free single-nucleus methods that may barcode nuclei more efficiently 67 . Second, Slide-tags is currently limited to single nucleus sequencing methods, primarily due to the ease of recovering nuclei from frozen tissues.…”

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

“…Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) have been similarly endowed with selective permeability and used to demonstrate enrichment of a sortase (employing a large particle sorter (COPAS, complex object parametric analyzer and sorter) instead of FACS) . Alternative materials provide routes to producing hydrogel beads as microspheres: alginate can be solidified with cations on-chip (Figure ) − or by laminar jetting into a bath, − and polyacrylamide can be cross-linked. , Beads based on hydrogels and other materials (e.g., polystyrene or paramagnetic composites) can also be used as a template to generate near-monodisperse droplets that tightly wrap around the bead via vortexing − or pipetting through filter tips , into an oil phase, avoiding microfluidic devices altogether.…”