Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing
Abstract
Single-cell analysis of gene expression is increasingly important for the analysis of complex tissues, including cancer, developing organs and adult stem cell niches. Here we present a detailed protocol for quantitative gene expression analysis in single cells, by the sequencing of mRNA 5' ends. In all, 96 cells are lysed, and their mRNA is converted to cDNA. By using a template-switching mechanism, a bar code and an upstream primer-binding sequence are introduced simultaneously with reverse transcription. All cDNA is pooled and then prepared for 5' end sequencing, including fragmentation, adapter ligation and PCR amplification. The chief advantage of this approach is the great reduction in cost and time, afforded by the early bar-coding strategy. Compared with previous methods, it is more suitable for large-scale quantitative analysis, as well as for the characterization of transcription start sites, but it is unsuitable for the detection of alternatively spliced transcripts. Sample preparation takes 3 d, and two sets of 96 cells can be prepared in parallel. Finally, the sequencing and data analysis can take an additional 4 d altogether.
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Many scRNA-seq approaches introduced over the last ten years are now key for measuring cell-to-cell variation and its effects on function across populations.
“Several scRNA-seq methods developed in the recent decade have become essential for capturing intercellular variation and its impact on cell functions at the population scale15–19.”
From The technological landscape and applications of single-cell multi-omics (2023) · cites this paper as [16]
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- Authors
- Islam, S., Kjällquist, U., Moliner, A., Zajac, P., Fan, J.-B., Lönnerberg, P., & Linnarsson, S.
- Affiliations
- Karolinska Institutet (SE)
- Illumina (United States) (US)
- Corresponding author
- Linnarsson, Sten
- Venue
- Nature Protocols
- Published
- 2012-04-05
- Volume
- 7
- Issue
- 5
- Pages
- 813-828
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2015329331
- Cited by
- 272
- References
- 27
- Citations by year
- 2026: 5 · 2025: 12 · 2024: 15 · 2023: 19 · 2022: 17 · 2021: 23 · 2020: 20 · 2019: 31 · 2018: 16 · 2017: 23 · 2016: 33 · 2015: 21 · 2014: 23 · 2013: 11 · 2012: 2
- FWCI
- 7.32
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- single-cell RNA sequencing, 5' end sequencing, strand-specific sequencing, template switching, reverse transcription, transcription start sites, gene expression quantification, single-cell transcriptomics
- MeSH terms
- Flow Cytometry, RNA, Messenger, Polymerase Chain Reaction, Sequence Analysis, RNA, DNA, Complementary, 5' Untranslated Regions, Nucleic Acid Amplification Techniques, Single-Cell Analysis
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