Abstract
The single-cell RNA-sequencing (scRNA-seq) field has evolved tremendously since the first paper was published back in 2009 (Tang et al. Nat Methods 6:377–382, 2009). While the first methods analyzed just a handful of cells, the throughput and performance rapidly increased over a very short time span. However, it was not until the introduction of emulsion droplets methods, such as the well-known kits commercialized by 10x Genomics, that the robust and reproducible analysis of thousands of cells became feasible (Zheng et al Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049, 2017). Despite generating data at a speed and a cost per cell that remains unmatched for full-length protocols like Smart-seq (Hagemann-Jensen et al Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38:708–714, 2020; Picelli et al Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096–1098, 2013), scRNA-seq in droplets still comes with the drawback of addressing only the terminal portion of the transcripts, thus lacking the required sensitivity for comprehensively analyzing the entire transcriptome.
Building upon the existing Smart-seq2/3 workflows (Hagemann-Jensen et al Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38:708–714, 2020; Picelli et al Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096–1098, 2013), we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than previous versions, requiring limited hands-on time and with a great potential for customization (Hahaut et al. Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq. http://biorxiv.org/lookup/doi/10.1101/2021.07.14.452217. https://doi.org/10.1101/2021.07.14.452217, 2021). Here, we present three variants of the FS protocol.
Standard FLASH-seq (FS), which builds upon Smart-seq2 developed in the past, is non-stranded and does not use unique molecular identifiers (UMIs) but still remains the easiest method to measure gene expression in a cell population.
FLASH-seq low-amplification (FS-LA) represents the fastest method, which generates sequencing-ready libraries in 4.5 h, without sacrificing performance.
FLASH-seq with UMIs (FS-UMI) builds upon the same principle as Smart-seq3 and introduces UMIs for molecule counting and isoform reconstruction. The newly designed template-switching oligonucleotide (TSO) contains a 5-bp spacer, which allows the generation of high-quality data while minimizing the amount of strand-invasion artifacts.
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Hahaut, V., Picelli, S. (2023). Full-Length Single-Cell RNA-Sequencing with FLASH-seq. In: Calogero, R.A., Benes, V. (eds) Single Cell Transcriptomics. Methods in Molecular Biology, vol 2584. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2756-3_5
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DOI: https://doi.org/10.1007/978-1-0716-2756-3_5
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