SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.
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.
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Tomonori
T
Nakamura
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Yukihiro
Y
Yabuta
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Ikuhiro
I
Okamoto
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Shinya
S
Aramaki
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan.
Shihori
S
Yokobayashi
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Kazuki
K
Kurimoto
Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
Kiyotoshi
K
Sekiguchi
Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan.
Masato
M
Nakagawa
Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Takuya
T
Yamamoto
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan saitou@anat2.med.kyoto-u.ac.jp.
Mitinori
M
Saitou
D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis