Smart-Seq/NanoCAGE/CAGEscan
Smart-Seq/NanoCAGE/CAGEscan
Switch mechanism at the 5’ End of RNA Templates, Smart-Seq , also known as NanoCAGE or CAGEscan, was developed as a single-cell sequencing protocol with improved read coverage across transcripts. Complete coverage across the genome allows the detection of alternative transcript isoforms and single-nucleotide polymorphisms. In this protocol, cells are lysed and the RNA hybridized to an oligo(dT)-containing primer. The first strand is then created with the addition of a few untemplated C nucleotides. This poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer is then hybridized to the poly(C) overhang and used to synthesize the second strand. Full-length cDNAs are PCR-amplified to obtain nanogram amounts of DNA. The PCR products are purified for sequencing.
- Smart-Seq: Ramskold D., Luo S., Wang Y. C., Li R., Deng Q., et al. (2012) Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol 30: 777-782
- NanoCAGE/CAGEscan: Plessy C., Bertin N., Takahashi H., Simone R., Salimullah M., et al. (2010) Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nat Methods 7: 528-534
- Agarwal S., Macfarlan T. S., Sartor M. A. and Iwase S. (2015) Sequencing of first-strand cDNA library reveals full-length transcriptomes. Nat Commun 6: 6002
- Kim D. H., Marinov G. K., Pepke S., Singer Z. S., He P., et al. (2015) Single-cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming. Cell Stem Cell 16: 88-101
- Tanaka Y., Hysolli E., Su J., Xiang Y., Kim K. Y., et al. (2015) Transcriptome Signature and Regulation in Human Somatic Cell Reprogramming. Stem Cell Reports 4: 1125-1139
- Bhargava V., Head S. R., Ordoukhanian P., Mercola M. and Subramaniam S. (2014) Technical variations in low-input RNA-seq methodologies. Sci Rep 4: 3678
- Biase F. H., Cao X. and Zhong S. (2014) Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res 24: 1787-1796