Small-seq for single-cell small-RNA sequencing

Nat Protoc. 2018 Oct;13(10):2407-2424. doi: 10.1038/s41596-018-0049-y.

Abstract

Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2-3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Complementary / genetics
  • Flow Cytometry / methods
  • Gene Library
  • HEK293 Cells
  • Humans
  • Polymerase Chain Reaction / methods
  • RNA, Small Untranslated / genetics*
  • RNA, Small Untranslated / isolation & purification
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*

Substances

  • DNA, Complementary
  • RNA, Small Untranslated