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Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] DOI:10.1002/0471142727.mb0422s107

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

Publication TypeJournal Article
Year of Publication2014
AuthorsTrombetta, JJ, Gennert, D, Lu, D, Satija, R, Shalek, AK, Regev, A
JournalCurrent protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
Date Published2014/07/01

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.