Single-cell semiconductor sequencing.
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.
The Whitney Laboratory for Marine Biosciences, University of Florida, Saint Augustine, FL, USA.
Andrea B
AB
Kohn
Tatiana P
TP
Moroz
Jeffrey P
JP
Barnes
Mandy
M
Netherton
Leonid L
LL
Moroz
D000375:Aging / Q000235:genetics; D000818:Animals; D001048:Aplysia / Q000196:embryology / Q000235:genetics*; D001483:Base Sequence; D020869:Gene Expression Profiling / Q000379:methods*; D016678:Genome / Q000235:genetics; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000166:cytology; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome / Q000235:genetics