Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing.
Curr Protoc Mol Biol
; 107: 4.22.1-4.22.17, 2014 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-24984854
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
Biblioteca Gênica
/
Análise de Sequência de RNA
/
Sequenciamento de Nucleotídeos em Larga Escala
Tipo de estudo:
Clinical_trials
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Curr Protoc Mol Biol
Ano de publicação:
2014
Tipo de documento:
Article