Single-Cell Semiconductor Sequencing
Open accessAbstract
RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field. It is especially critical for developmental, aging, and cancer biology as well as neuroscience where the enormous heterogeneity of cells present a significant methodological and conceptual challenge. Here we present two methods that allow for fast and cost-efficient transcriptome sequencing from ultra-small amounts of tissue or even from individual cells using semiconductor sequencing technology (Ion Torrent, Life Technologies). The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts' directionality. The second, a template-switch protocol, is designed for small mammalian neurons. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The efficiency of these protocols has been validated with single hippocampal neurons and various invertebrate tissues including individually identified neurons within a simpler memory-forming circuit of Aplysia californica and early (1-, 2-, 4-, 8-cells) embryonic and developmental stages from basal metazoans.
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A reduced representation sequencing protocol for 454 and Ion Torrent platforms can detect low abundance transcripts, reduces the amount of sequencing, and gives more accurate quantification.
“We developed a reduced representation sequencing protocol for the 454 and Ion Torrent sequencing platforms that can detect low abundance transcripts 47 . The method reduces the amount of sequencing and gives more accurate quantification and additional details of the procedure are reported elsewhere 47,48 .”
From The ctenophore genome and the evolutionary origins of neural systems (2014) · cites this paper as [47]
The Introduction and Methods §3.3 describe a reduced-representation protocol for Ion Torrent; §3.3 says it captures more distinct transcripts with less sequencing coverage and is designed for quantitative mRNA analysis. Our copy does not establish that the protocol is for 454, specifically detects low-abundance transcripts, or gives more accurate quantification than another method. (LLM, conf=0.83)
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The ctenophore genome and the evolutionary origins of neural systems
Moroz, L. L., Kocot, K. M., Citarella, M. R., Dosung, S., Norekian, T. P., Povolotskaya, I. S., Grigorenko, A. P., Dailey, C., Berezikov, E., Buckley, K. M., et al. (2014) · Nature · doi:10.1038/nature13400 · Cited by 716 · see citing works
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Single-Neuron Transcriptome and Methylome Sequencing for Epigenomic Analysis of Aging
Moroz, L. L., & Kohn, A. B. (2013) · Methods in Molecular Biology · doi:10.1007/978-1-62703-556-9_21 · Cited by 42 · see citing works
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- Authors
- Kohn, A. B., Moroz, T. P., Barnes, J. P., Netherton, M., & Moroz, L. L.
- Affiliations
- University of Florida (US)
- Venue
- Methods in Molecular Biology
- Publisher
- Humana Press
- Published
- 2013
- Volume
- 1048
- Pages
- 247-284
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- PMC
- PMC4024476
- OpenAlex
- W166289607
- Cited by
- 22
- References
- 26
- Citations by year
- 2024: 1 · 2023: 1 · 2021: 3 · 2020: 1 · 2019: 1 · 2018: 3 · 2017: 1 · 2016: 4 · 2015: 4 · 2014: 2 · 2013: 1
- FWCI
- 4.80
- Type
- book chapter
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation
- National Institutes of Health
- National Institute of Mental Health (R01MH097062)
- National Institute on Drug Abuse (R21DA030118)
- National Institute of General Medical Sciences (R01GM097502)
- National Center for Research Resources (R21RR025699)
- 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 transcriptomics, semiconductor sequencing, Ion Torrent, reduced representation sequencing, hippocampal neurons, Aplysia californica, embryonic development, invertebrate development, RNA capture
- MeSH terms
- Aging, Animals, Aplysia, Base Sequence, Hippocampus, Neurons, Genome, Sequence Analysis, RNA, Gene Expression Profiling, Transcriptome
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