Smart-Seq2
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 significantly improve the yield. In this protocol, single cells are lysed in a buffer that contains free dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5′-anchor sequence. Reverse transcription is performed, which adds 2–5 untemplated nucleotides to the cDNA 3′ end. A template-switching oligo (TSO) is added, carrying two riboguanosines and a modified guanosine to produce a LNA as the last base at the 3′ end. After the first-strand reaction, the cDNA is amplified using a limited number of cycles. Tagmentation is then used to quickly and efficiently construct sequencing libraries from the amplified cDNA.
Pros:
- The sequence of the mRNA does not have to be known
- As little as 50 pg of starting material can be used
- Improves coverage across transcripts
- High level of mappable reads
Cons:
- Not strand-specific
- No early multiplexing
- Applicable only to poly(A)+ RNA
- Smart-Seq2: Picelli S., Bjorklund A. K., Faridani O. R., Sagasser S., Winberg G., et al. (2013) Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10: 1096-1098
- Smart-Seq2: Picelli S., Faridani O. R., Björklund Å. K., Winberg G., Sagasser S., et al. (2014) Full-length RNA-Seq from single cells using Smart-seq2. Nat. Protocols 9: 171-181
- Macaulay I. C., Haerty W., Kumar P., Li Y. I., Hu T. X., et al. (2015) G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods 12: 519-522
- Moignard V., Woodhouse S., Haghverdi L., Lilly A. J., Tanaka Y., et al. (2015) Decoding the regulatory network of early blood development from single-cell gene expression measurements. Nat Biotechnol 33: 269-276
- Swiech L., Heidenreich M., Banerjee A., Habib N., Li Y., et al. (2015) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33: 102-106
- Deng Q., Ramskold D., Reinius B. and Sandberg R. (2014) Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343: 193-196
- Picelli S., Faridani O. R., Bjorklund A. K., Winberg G., Sagasser S., et al. (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9: 171-181