RESUMO
Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
Assuntos
Blastômeros/metabolismo , Perfilação da Expressão Gênica/métodos , Oócitos/metabolismo , Análise de Sequência de DNA/métodos , Algoritmos , Animais , Proteínas Argonautas , Blastômeros/citologia , Ciclina E/genética , RNA Helicases DEAD-box/genética , DNA Complementar/síntese química , DNA Complementar/genética , Bases de Dados de Ácidos Nucleicos , Endorribonucleases/genética , Fator de Iniciação 2 em Eucariotos/genética , Feminino , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas/genética , Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ribonuclease III , Alinhamento de Sequência , Regulação para Cima/genéticaRESUMO
We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Animais , Sequência de Bases , Blastômeros/metabolismo , DNA Complementar/genética , Feminino , Biblioteca Gênica , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oócitos/metabolismo , RNA/genética , RNA/isolamento & purificaçãoRESUMO
We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.