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. 2009 Dec;20(5):266-71.

mRNA-sequencing whole transcriptome analysis of a single cell on the SOLiD system

Kai Q Lao  1 , Fuchou Tang, Catalin Barbacioru, Yangzhou Wang, Ellen Nordman, Clarence Lee, Nanlan Xu, Xiaohui Wang, Brain Tuch, John Bodeau, Asim Siddiqui, M Azim Surani
Affiliations

mRNA-sequencing whole transcriptome analysis of a single cell on the SOLiD system

Kai Q Lao et al. J Biomol Tech. 2009 Dec.

Abstract

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.

Keywords: Ago2; Dicer; gene expression.

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Figures

FIGURE 1
FIGURE 1
Reaction schemes. UP1, universal primer 1; UP2, universal primer 2.
FIGURE 2
FIGURE 2
cDNA products. KO, Knockout.
FIGURE 3
FIGURE 3
SOLiD technology.
FIGURE 4
FIGURE 4
SOLiD matching summary for mouse blastomeres in four-cell embryos.
FIGURE 5
FIGURE 5
SOLiD results for wild-type (Wt) and Dicer-KO oocytes.
FIGURE 6
FIGURE 6
Comparison of SOLiD sequencing data with those from microarrays of ∼80 pooled four-cell stage embryos (320 blastomeres) found that 6733 genes detected by Affymetrix GeneChip Mouse Genome 430 2.0 array (Affy Array) were also detected by SOLiD sequencing. The 317 genes, detected by microarray but missed by SOLiD, were relatively low-expressed genes and could result from cross-hybridization. SOLiD sequencing detects 4877 more expressed genes compared with microarray.
FIGURE 7
FIGURE 7
Coverage of exons for Pou5f1 (Oct4).
FIGURE 8
FIGURE 8
Dicer locus on Chromosome 12 and coverage of Dicer Exon 23. LoxP, Locus of crossover in P1.
FIGURE 9
FIGURE 9
The correlation plots of the fold changes that are determined by SOLiD reads and real-time PCR. FC, fold changes.
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