Abstract
Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.
Highlights
Quantitative transcriptome analysis at single-cell resolution is becoming an increasingly important area of biomedical sciences, including in the research fields of developmental/stem cell/cancer biology, and is providing a foundation for understanding the regulation of gene expression in single cells in physiology or diseased states at a systems level [1,2]
At E4.5, peri-implantation blastocysts were flushed from the uteri by KSOM [Merck Millipore (MR-020P-5D), Darmstadt, Germany], and they were bisected into a polar part containing an inner cell mass (ICM) and polar trophectoderm and a mural part containing mural TE by a glass needle under a dissection microscope [Leica Microsystems (M80), Wetzlar, Germany]
We have previously reported a method for the amplification of single-cell cDNAs enriched in the 3-prime ends (687 ± 279 bp) for high-density oligonucleotide microarray analysis [7,8]
Summary
Quantitative transcriptome analysis at single-cell resolution is becoming an increasingly important area of biomedical sciences, including in the research fields of developmental/stem cell/cancer biology, and is providing a foundation for understanding the regulation of gene expression in single cells in physiology or diseased states at a systems level [1,2]. The methods involving exponential amplification have higher amplification efficiency, greater methodological simplicity and higher stability of the amplified products, which allows an examination of the amplification quality prior to global measurements/repeated assessment of the same single-cell transcriptomes. To ensure quantitative/representative amplification of single-cell cDNAs, one of the original methods that applied amplified cDNAs to global analyses using highdensity oligonucleotide microarrays restricted the length of the first-strand cDNAs to, on average, ∼700 base pairs (bp) from the 3-prime ends [transcription termination sites (TTSs)] of mRNAs, by a short (5 min) reverse transcription [7,8].
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