Full-Length Single-Cell RNA Sequencing with Smart-seq2
In the last few years single-cell RNA sequencing (scRNA-seq) has enabled the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types as well as the detailed analysis of the stochastic nature of gene ex
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Introduction The rapid technological development of the single-cell sequencing field in the past 10 years has enabled researchers to answer questions that could not be addressed by the classic RNA-seq or microarray technologies. It is now well established that seemingly homogenous cell populations in vivo and cell cultures in vitro can display considerable differences in gene expression, both due to stochastic processes at the transcriptional level (e.g., transcriptional burst) or extrinsic factors such as experimental conditions [1–3]. Among all the different applications developed over the years, single-cell RNA sequencing (scRNA-seq) is the one that has seen major improvements, both in terms of sensitivity and scalability. Some of the technologies, including the latest emulsion droplet methods like Drop-seq, inDrop, and the 10 Genomics technology [4–6], characterize only the 30 -end of the RNA transcripts, which is generally sufficient for the investigation of cellular heterogeneity and the identification of population substructures. Other methods such as Smart-seq2, SUPeR-seq, and MATQ-seq [7–9]
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_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Simone Picelli
Fig. 1 Flowchart of the Smart-seq2 library preparation. Single cells are collected manually or by FACS and deposited in single tubes or 96-/384-well plates containing a mild hypotonic lysis buffer. The cells are lysed and the RNA is released. The RT reaction begins with the annealing of an oligo-dT primer (SMART dT30VN)
Single-Cell RNA-seq with Smart-seq2
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have the ability to capture full-length transcripts and are therefore useful for the characterization of single nucleotide variants (SNVs), splice isoform, and transcriptional start sites (TSSs), or for the detection of monoallelic and imprinted genes. Smart-seq2 relies on the SMART technology (Switching Mechanism at the 50 -end of the RNA Transcript) and exploits two intrinsic properties of the Moloney murine leukaemia virus reverse transcriptase (MMLV-RT): reverse transcription (RT) and template switching (TS) [10]. Template switching represents the ability of the MMLV-RT to introduce a few untemplated nucleotides, most commonly 2–5 cytosines, upon reaching the 50 -end of the RNA template during the RT reaction (Fig. 1). These extra nucleotides work as a docking site for a helper oligonucleotide (“template switching oligonucleotide,” TSO) carrying two riboguanosines and one locked nucleic acid (LNA) guanosine at its 30 -end. This special base configuration is crucial for a stable annealing between the TSO and the cytosine tail and is required for the MMLV-RT to “switch template” and synthesize a cDNA strand using the helper oligonucleotide as template. Thus, TS makes possible the introduction of a predefined sequence at the 30 -end end of the cDNA transcript (the 50 -end of the mRNA template) which, notably,
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