UNIT 4.22 Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing

  1. John J. Trombetta1,
  2. David Gennert1,
  3. Diana Lu1,
  4. Rahul Satija1,
  5. Alex K. Shalek2,
  6. Aviv Regev1,3

Published Online: 1 JUL 2014

DOI: 10.1002/0471142727.mb0422s107

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Trombetta, J. J., Gennert, D., Lu, D., Satija, R., Shalek, A. K. and Regev, A. 2014. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. Current Protocols in Molecular Biology. 107:II:4.22:4.22.1–4.22.17.

Author Information

  1. 1

    Broad Institute of MIT and Harvard, Cambridge, Massachusetts

  2. 2

    Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, Massachusetts

  3. 3

    Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

Publication History

  1. Published Online: 1 JUL 2014


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. Curr. Protoc. Mol. Biol. 107:4.22.1-4.22.17. © 2014 by John Wiley & Sons, Inc.


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