Smart-Seq/NanoCAGE/CAGEscan

Smart-Seq/NanoCAGE/CAGEscan

Smart-Seq and Smart-Seq2: Switch Mechanism at the 5' End of RNA Templates Smart-Seq 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 SNPs. There are 2 versions of Smart-Seq: Smart-Seq and Smart-seq2. Smart-seq2 includes several improvements over the original Smart-Seq protocol. The new protocol includes a locked nucleic acid (LNA), an increased MgCl2 concentration, betaine, and elimination of the purification step to improve the yield significantly.

Smart-Seq: Cells are lysed, and the RNA is hybridized to an oligo(dT)-containing primer. The first strand of the cDNA is synthesized with the addition of a few untemplated C nucleotides. This poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer is 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.

Pros:

• mRNA sequence does not have to be known
• As little as 50 pg of starting material can be used
• Improved coverage across transcripts
• High level of mappable reads

Cons:

• Not strand-specific
• No early multiplexing
• Applicable only to poly(A)+ RNA

• 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

• Smart-Seq: Ramskold D., Luo S., Wang Y. C., et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol. 2012;30:777-782

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