Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.

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Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.[Supplemental material is available for this article.]The unprecedented increase in sequencing machine capacity (Metzker 2009) has led to improved throughput and lowered cost for DNA and RNA sequencing (RNA-seq) applications. For example, expression profiles from hundreds of different single cells can be generated in a single lane of an Illumina HiSeq 2000 (Hashimshony et al. 2012;Islam et al. 2014;Jaitin et al. 2014). While the sequencing costs have been drastically decreasing, the throughput and costs of library preparation are now the limiting factor. This represents a severe constraint in many next-generation sequencing-based projects, especially now that the importance of obtaining expression profiles from hundreds or thousands of single cells is more fully appreciated (Eberwine et al. 2014;Sandberg 2014). Indeed, improved automation and lowered reagent costs in sequence library generation are needed to harness the full potential of current sequencing technology and to make it affordable for any laboratory around the world.A great advancement in library preparation was the introduction of a hyperactive variant of the Tn5 transposase that mediates the fragmentation of double-stranded DNA and ligates synthetic oligonucleotides at both ends in a 5-min reaction (Adey et al. 2010). Wild-type Tn5 transposon is a composite transposon in which two near-identical insertion sequences (IS50L and IS50R) are flanking three antibiotic resistance genes (Reznikoff 2008). Each IS50 contains two inverted 19-bp end sequences (ESs), an outside end (OE) and an inside end (IE). However, wild-type ESs have a relatively low activity and were replaced in vitro by hyperactive mosaic end (ME) sequences. A complex of the transposase with the 19-bp ME is thus all that is necessary for transposition to occur, provided that the inte...