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

J. J. Trombetta, D. Gennert, D. Lu, R. Satija, A. K. Shalek, A. Regev

Research output: Contribution to journalArticle

Abstract

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. (c) 2014 by John Wiley & Sons, Inc.
Original languageUndefined
Pages (from-to)4.22.1-4.22.17
JournalCurr Protoc Mol Biol
Volume107
DOIs
StatePublished - 2014

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