Highly parallel and efficient single cell mRNA sequencing with paired picoliter chambers

Abstract

ScRNA-seq has the ability to reveal accurate and precise cell types and states. Existing scRNA-seq platforms utilize bead-based technologies uniquely barcoding individual cells, facing practical challenges for precious samples with limited cell number. Here, we present a scRNA-seq platform, named Paired-seq, with high cells/beads utilization efficiency, cell-free RNAs removal capability, high gene detection ability and low cost. We utilize the differential flow resistance principle to achieve single cell/barcoded bead pairing with high cell utilization efficiency (95%). The integration of valves and pumps enables the complete removal of cellfree RNAs, efficient cell lysis and mRNA capture, achieving highest mRNA detection accuracy (R = 0.955) and comparable sensitivity. Lower reaction volume and higher mRNA capture and barcoding efficiency significantly reduce the cost of reagents and sequencing. The singlecell expression profile of mES and drug treated cells reveal cell heterogeneity, demonstrating the enormous potential of Paired-seq for cell biology, developmental biology and precision medicine.

Deterministic scRNA-seq of individual intestinal organoids reveals new subtypes and coexisting distinct stem cell pools

“…This enables the correct assembly of most droplets, virtually eradicating confounding factors that arise due to failed coencapsulations 37,38 . In concept, DisCo is thus fundamentally different to passive particle pairing approaches such as traps [39][40][41] and, compared to these technologies, offers the advantage of requiring vastly simpler and reusable chips without suffering from cell/particle size and shape selection biases 13,42 . This renders the DisCo approach universally applicable to any particle coencapsulation application 43,44 , i.e.…”

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing

“…This reduces the problem to a single Poisson statistic allowing the majority of cells to be captured, albeit necessitating lower droplet generation rates, ultimately producing equivalent throughput to solid bead systems. The general problem of single cell encapsulation has attracted great interest, resulting in a wide variety of passive (trapping, 12,13 deterministic 14 and inertial ordering [15][16][17] ) and active (electrical, [18][19][20] magnetic, 21 optical, 22 acoustic 23 and mechanical 24 ) droplet generation and encapsulation strategies that have recently been expertly reviewed by Ling et al 25 Passive approaches harness flow and channel properties, with the benefit of being easy to operate. In particular, inertial microfluidic formats [26][27][28] enable high throughput and can produce entrainment effects to offer the enticing possibility of the periodic delivery of cell and beads, allowing deterministic packaging into droplets to free assays from the limitations of the Poisson statistic.…”

Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics

“…However, these droplet-based approaches still face various difficulties to completely fulfill WGA requirements. First, the strategy of single-cell isolation based on Poisson statistics causes low cell occupancy and high loss of cells, which is inaccessible for rare samples ( 31 ). Moreover, these approaches are hard to manipulate and to control the droplets addressably in parallel, which limit the capability of picking up desirable droplets.…”