Seq-Well: A Sample-Efficient, Portable Picowell Platform for Massively Parallel Single-Cell RNA Sequencing
Seq-Well is a low-cost picowell platform that can be used to simultaneously profile the transcriptomes of thousands of cells from diverse, low input clinical samples. In Seq-Well, uniquely barcoded mRNA capture beads and cells are co-confined in picowells
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Introduction Single-cell RNA sequencing (scRNA-seq) is an emerging method that enables genome-wide expression profiling at cellular resolution. Population-level transcriptomic techniques, such as microarrays and bulk RNA-seq, average over a large number of cells and assume transcriptional homogeneity; yet even related cells of the same subtype can present dramatic heterogeneity in their transcriptional activities and states [1]. ScRNA-seq allows direct measurement of this variability, as well as analyses of expression covariation across cells. This information can be used to discover gene-expression patterns that define distinct cell types and states, as well as their molecular circuits and biomarkers, affording an unprecedented view of cellular phenotype. Over the years, technological progress and protocol improvements have resulted in a substantial increase
Junior authors, Toby P. Aicher, Shaina Carroll, and Gianmarco Raddi, and senior authors, Chris Love and Alex K. Shalek, contributed equally to this work. Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_8, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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in the number of cells that can be processed in parallel [2–4], enhancing statistical power and providing opportunities to look at increasingly complex systems. Current methods used to prepare single-cell libraries include manual selection [5], FACS sorting [6], microfluidic circuits [7], droplet-based techniques [8–10], and picowells [11, 12]. Seq-Well, an example of the latter, is an easy-to-use, low-cost, sample-efficient and portable platform for massively parallel scRNA-seq [11]. Seq-Well utilizes PDMS arrays containing ~88,000 subnanoliter wells in which single cells and uniquely barcoded poly(dT) mRNA beads are co-confined with a semipermeable membrane. Crucially, well size ensures that only one barcoded mRNA capture bead can fit into each well, improving cell capture efficiency. Cells, meanwhile, are loaded at a low density to minimize cell doublets, ensuring single-cell resolution. Selective chemical functionalization allows reversible attachment of a semipermeable polycarbonate membrane with 10 nm pores, permitting buffer exchange for cell lysis while trapping larger macromolecules, such as nucleic acids, to minimize cross-contamination. The co-confined mRNA capture beads are covered in oligonucleotides that consist of a universal primer, a cell barcode (unique to each bead), a unique molecular identifier (UMI; unique to each primer), and a poly-T sequence that can capture cellular mRNA upon lysis and during hybridization [13]. Following these steps, the semipermeable membrane can be peeled off for bead removal. Finally, the barcoded beads can be pooled for reverse transcription, PCR amplification, library preparation, and sequencing, with a transcript’s cell of origin and uniqueness determined via its cell barcode and UMI, respectively.
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