Dean flow assisted single cell and bead encapsulation for high performance single cell expression profiling

Abstract

Single-cell RNA sequencing examines the transcriptome of individual cells and reveals the inter-cell transcription heterogeneity, playing a critical role in both scientific research and clinical applications. Recently, droplet microfluidics-based platform for expression profiling has been shown as a powerful tool to capture of the transcriptional information on single cell level. Despite the breakthrough this platform brought about, it required the simultaneous encapsulation of single cell and single barcoded bead, the incidence of which was very low. Suboptimal capturing efficiency limited the throughput of the Drop-seq platform. In this work, we leveraged the advance in inertial microfluidics-based cell sorting and designed a microfluidic chip for high efficiency cell-bead co-encapsulation, increasing the capturing rate by more than four folds.Specifically, we adopted spiral and serpentine channels and ordered cells/beads before the encapsulation region. We characterized the effect of cell concentration on the capturing rate and achieved a cell-bead co-capturing rate up to 3%. We tested this platform by co-encapsulating barcoded beads and human-mouse cell mixtures. The sequencing data distinguished the majority of human and mice expressions, with the doublet rate being as low as 5.8%, indicating that the simultaneous capturing of two or more cells in one droplet was minimal even when using high cell concentration. This chip design showed great potential in improving the efficiency for future single cell expression profiling.

Dean Flow Assisted Single Cell and Bead Encapsulation for High Performance Single Cell Expression Profiling

“…The copyright holder for this preprint (which was not this version posted April 29, 2019. ; https://doi.org/10.1101/520858 doi: bioRxiv preprint Overall, qualified library was built after completion of DNA fragmentation and cyclization. We used dsDNA fragmentase (M0348L, NEB) to interrupt the target sequence (cDNA) randomly, followed by end-repairing and adding adapter primers 18 .…”

Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics

“…Chemical lysis is simple to use, in some cases it only requires a short period of heating for a better lysis performance. And chemical lysis is applicable to any microfluidic platforms for single-cell nucleic acid analysis (Wood et al 2010 ; VanInsberghe et al 2018 ; Li et al 2019a ). However, chemical lysis buffer may inhibit the subsequent reaction, so after cell lysis it is necessary to remove residual lysis buffer in the chamber or neutralized by other reagents.…”