Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing

Curr Protoc Mol Biol. 2014 Jul 1;107:4.22.1-17. doi: 10.1002/0471142727.mb0422s107.


For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second "template-switch" primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.

Keywords: Next-generation sequencing; SMART-Seq; SMART-Seq2; cell type; low-input RNA-Seq; single cell; single-cell RNA-Seq; template-switching; transcriptomics.

Publication types

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

MeSH terms

  • Animals
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • Gene Library*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • RNA, Messenger* / chemistry
  • RNA, Messenger* / genetics
  • RNA, Messenger* / isolation & purification
  • Sequence Analysis, RNA / methods*


  • DNA, Complementary
  • RNA, Messenger