The current trends are to adapt high-throughput sequencing technologies to the level of small-cell populations and even individual cells.
It has become indispensable for virtually any biomedical field, providing an unbiased view of the entire molecular machinery within a biological system of interest. 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.ĭNA and RNA sequencing has and will continue to make a profound impact on medicine, clinical, and basic research. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The second, a template-switch protocol, is designed for small mammalian neurons. The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts’ directionality. 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). 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. RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field.