Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters

Anal Chem. 2018 Nov 6;90(21):12609-12615. doi: 10.1021/acs.analchem.8b02773. Epub 2018 Sep 27.

Abstract

Although single-cell mRNA sequencing has been a powerful tool to explore cellular heterogeneity, the sequencing of small RNA at the single-cell level (sc-sRNA-seq) remains a challenge, as these have no consensus sequence, are relatively low abundant, and are difficult to amplify in a bias-free fashion. We present two methods of single-cell-lysis that enable sc-sRNA-seq. The first method is a chemical-based technique with overnight freezing while the second method leverages on-chip electrical lysis of plasma membrane and physical extraction and separation of cytoplasmic RNA via isotachophoresis. We coupled these two methods with off-chip small RNA library preparation using CleanTag modified adapters to prevent the formation of adapter dimers. We then demonstrated sc-sRNA-seq with single K562 human leukemic cells. Our approaches offer a relatively short hands-on time of 6 h and efficient generation of on-target reads. The sc-sRNA-seq with our approaches showed detection of miRNA with various abundances ranging from 16 000 copies/cell to about 10 copies/cell. We anticipate this approach will create a new opportunity to explore cellular heterogeneity through small RNA expression.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Genetic Structures
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • K562 Cells
  • Lab-On-A-Chip Devices
  • Octoxynol / chemistry
  • RNA, Small Untranslated / genetics*
  • RNA, Small Untranslated / isolation & purification
  • Reproducibility of Results
  • Sequence Analysis, RNA
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods*

Substances

  • RNA, Small Untranslated
  • Octoxynol