Single-Cell RNA Sequencing with Drop-Seq
Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture microparticles and cells are coconfined through a microfluidic device within the droplets where t
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Introduction Single-cell RNA-seq allows biological investigators to tap the transcriptome of individual cells to analyze which cell types they belong to, what their lineage is, and what transcriptional modules they possess [1, 2]. In a short amount of time, single-cell RNA-seq (scRNA-seq) has become a popular tool used in all branches of biology [3]. Protocol improvements and technological advancements have led to an exponential growth in the number of cells being analyzed, allowing in turn for the discovery of even rarer subtypes [3, 4]. Manual selection [5], FACS sorting [6], and microfluidic circuits led the way [7], but it was only with dropletbased techniques such as Drop-seq that thousands of individual cells could be routinely profiled in each experiment [8–10]. Drop-seq involves coencapsulation of single cells and barcoded microparticles in nanoliter-sized droplets [9]. Cells and beads are strongly diluted such that only a few droplets will contain a bead or a cell, or both, enabling a very low doublet rate but resulting also in a lower cell capture efficiency (number of captured cells/number of cells used). Once cells are encapsulated within the droplet, they are
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_6, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Josephine Bageritz and Gianmarco Raddi
immediately lysed which causes the release of polyadenylated mRNA transcripts. Breaking the droplets and reverse transcription of their mRNA forms covalent and stable STAMPs (single-cell transcriptomes attached to microparticles). Exonuclease treatment is then applied to remove bead primers that have not captured an mRNA molecule, after which cDNA amplification, library construction, and sequencing are performed [9]. This droplet-based microfluidics technology can be set up in a cost-effective manner in each laboratory. Droplet technologies in general cut the cost of library preparation per cell tenfold compared to FACS approaches such as Smartseq2. However, only the 30 -end of the transcript can be sequenced, thus losing information that alternative full-coverage techniques can provide [1]. All in all, Drop-seq is a flexible and cost-effective method that allows many laboratories to assess the transcriptome of single cells on a large scale. Furthermore, it can easily be modified to suit individual needs (DroNcSeq [11], CITE-Seq [12]).
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Materials
2.1 Cell Encapsulation and STAMP Generation
It is recommended to prepare all buffers/reagents using ultrapure water and molecular biological grad reagents. Store them at room temperature, unless indicated otherwise. 1. Three infuse only syringe pumps (e.g., KD Scientific Legato 100). 2. Inverted microscope. 3. Multi Stirrus magnetic tumble stirrer (V&P Scientific). 4. Accessory kit for positioning the Multi Stirrus (V&P Scientific). 5. Mixing disc (e.g., V&P Scientific). 6. Aquapel-coated PDMS microfluidics device (e.g.,
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