Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells
Open accessAbstract
Genome-wide transcriptome analyses are routinely used to monitor tissue-, disease- and cell type–specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which enhances detailed analyses of alternative transcript isoforms and identification of single-nucleotide polymorphisms. We determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. We found that although gene expression estimates from single cells have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type. Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including candidate biomarkers for melanoma circulating tumor cells. Our protocol will be useful for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.
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Newer protocols such as Smart-seq and Smart-seq2 have enabled work from very small amounts of starting mRNA that can be obtained from just a single cell.
“Newer protocols such as Smart-seq [173] and Smart-seq2 [174] have enabled us to work from very small amounts of starting mRNA that, with proper amplification, can be obtained from just a single cell.”
From A survey of best practices for RNA-seq data analysis (2016) · cites this paper as [173]
Rapid technological advances have enabled genome-wide profiling of RNA, DNA, protein, epigenetic modifications, chromatin accessibility, and other molecular events in single cells.
“rapid technological advances have enabled genome-wide profiling of RNA7–10, DNA11–17, protein18–21, epigenetic modifications22–26, chromatin accessibility27,28, and other molecular events29 in single cells.”
From Revealing the vectors of cellular identity with single-cell genomics (2016) · cites this paper as [8]
Limited efficiency of RNA capture and conversion into cDNA leads to prevalent dropout events (false negatives) in scRNA-seq.
“At the other end of the spectrum, the limited efficiency of RNA capture and conversion into cDNA leads to prevalent dropout events (false negatives): transcripts that are expressed in the cell but are entirely undetected in its mRNA profile8 (Fig. 3c).”
From Revealing the vectors of cellular identity with single-cell genomics (2016) · cites this paper as [8]
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 [18]
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- Authors
- Ramsköld, D., Luo, S., Wang, Y.-C., Li, R., Deng, Q., Faridani, O. R., Daniels, G. A., Khrebtukova, I., Loring, J. F., Laurent, L. C., Schroth, G. P., & Sandberg, R.
- Affiliations
- Ludwig Cancer Research (SE)
- Illumina (United States) (US)
- Scripps Research Institute (US)
- Moores Cancer Center (US)
- University of California San Diego (US)
- Karolinska Institutet (SE)
- Corresponding author
- Sandberg, Rickard
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2012-07-22
- Volume
- 30
- Issue
- 8
- Pages
- 777-782
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3467340
- OpenAlex
- W2121690903
- Cited by
- 1,609
- References
- 35
- Citations by year
- 2026: 63 · 2025: 106 · 2024: 103 · 2023: 108 · 2022: 138 · 2021: 134 · 2020: 153 · 2019: 145 · 2018: 127 · 2017: 121 · 2016: 113 · 2015: 105 · 2014: 110 · 2013: 63 · 2012: 17
- FWCI
- 37.69
- Type
- journal article
- Language
- en
- Update notices
- correction (2020-02-03)
- Open access
- green
- europepmc.org
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Mental Health (R33MH087925)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (K12HD001259)
- 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, SMART-seq, alternative splicing, single nucleotide polymorphisms, stochastic gene expression, circulating tumor cells, melanoma, biomarkers
- MeSH terms
- Animals, Female, Humans, Melanoma, Neoplastic Cells, Circulating, RNA, Messenger, Sensitivity and Specificity, Gene Library, Cluster Analysis, Sequence Analysis, RNA, Oligonucleotide Array Sequence Analysis, Gene Expression Profiling, Mice
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