Abstract
Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.
Highlights
When dealing with specimens containing a small number of cells such as core biopsies or subpopulations isolated by flow sorting or laser capture microdissection, cell suspensions loaded into the device can be agitated with laminar flow to increase the fraction of cells captured by the microwell array
The 5′-ends of the oligo(dT) primers contain a universal adapter sequence for amplification, a barcode sequence that is unique to the bead, and a second barcode sequence for uniquely labeling a captured cDNA molecule[7,20,21]
We have demonstrated single cell RNA-Seq in PDMS microwells using capture beads that contain specific barcode sequences generated by combinatorial primer extension[9] and, in this report, using the random barcodes sequences generated by split-pool solid phase oligonucleotide synthesis[7]
Summary
We have demonstrated single cell RNA-Seq in PDMS microwells using capture beads that contain specific barcode sequences generated by combinatorial primer extension[9] and, in this report, using the random barcodes sequences generated by split-pool solid phase oligonucleotide synthesis[7]. On-chip fluorescence imaging facilitates quality-control of cell viability and lysis and microwell sealing quality while providing a simple means of counting the number of cell-bead pairs and multiplet loading rate in every experiment.
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