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

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

Author information

1
Broad Institute of MIT and Harvard, Cambridge, Massachusetts.

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.

KEYWORDS:

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

PMID:
24984854
PMCID:
PMC4338574
DOI:
10.1002/0471142727.mb0422s107
[Indexed for MEDLINE]
Free PMC Article

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