Selective single cell isolation for genomics using microraft arrays

Nucleic Acids Res. 2016 Sep 30;44(17):8292-301. doi: 10.1093/nar/gkw700. Epub 2016 Aug 16.

Abstract

Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.

Publication types

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

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Separation / methods*
  • Cells, Cultured
  • Deoxycytidine / analogs & derivatives
  • Deoxycytidine / pharmacology
  • Gene Expression Regulation, Neoplastic / drug effects
  • Genomics / methods*
  • Humans
  • Mice
  • Microarray Analysis*
  • Reproducibility of Results
  • Sequence Analysis, RNA
  • Tumor Stem Cell Assay

Substances

  • Deoxycytidine
  • gemcitabine