Single-cell semiconductor sequencing

Methods Mol Biol. 2013;1048:247-84. doi: 10.1007/978-1-62703-556-9_18.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aging / genetics
  • Animals
  • Aplysia / embryology
  • Aplysia / genetics*
  • Base Sequence
  • Gene Expression Profiling / methods*
  • Genome / genetics
  • Hippocampus / cytology
  • Neurons / cytology
  • Sequence Analysis, RNA / methods*
  • Transcriptome / genetics