Single-Cell mRNA-Seq Using the Fluidigm C1 System and Integrated Fluidics Circuits

Methods Mol Biol. 2018:1783:193-207. doi: 10.1007/978-1-4939-7834-2_10.

Abstract

Single-cell mRNA-seq is a valuable tool to dissect expression profiles and to understand the regulatory network of genes. Microfluidics is well suited for single-cell analysis owing both to the small volume of the reaction chambers and easiness of automation. Here we describe the workflow of single-cell mRNA-seq using C1 IFC, which can isolate and process up to 96 cells. Both on-chip procedure (lysis, reverse transcription, and preamplification PCR) and off-chip sequencing library preparation protocols are described. The workflow generates full-length mRNA information, which is more valuable compared to 3' end counting method for many applications.

Keywords: Gene expression; Integrated fluidic circuit; Single-cell; mRNA-seq.

MeSH terms

  • DNA, Complementary / biosynthesis
  • DNA, Complementary / genetics
  • Gene Expression Profiling / methods*
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Microfluidic Analytical Techniques / instrumentation*
  • Microfluidic Analytical Techniques / methods
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Transcriptome*

Substances

  • DNA, Complementary
  • RNA, Messenger