Summary of single cell RNA-sequencing technology. Please let me know if there is another methods or if you find some misunderstanding.
a) Smart-seq2 & Smart-seq (= the SMARTer Ultra Low RNA Kit from Clontech)
By reverse-transcription with oligo-dT anchored primer with reverse-transcriptase from the Moloney murine leukemia virus (M-MLV RT), a few non-templated cytosines are added. After second strand synthesis using oligo-dG primer, amplification of dsDNA is performed by PCR. Smart-seq2 is optimized method of Smart-seq, so the basic technology used for library construction is same as previous version. Sensitivity, accuracy and full-length coverage of transcripts are improved.
1. Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nature protocols 9, 171-181, doi:10.1038/nprot.2014.006 (2014). (published from Karolinska Institutet)
2. Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature biotechnology 30, 777-782, doi:10.1038/nbt.2282 (2012). (published from Karolinska Institutet)
After reverse-transcription with oligo-dT anchored primer, poly-A is added to the 3′ end of the first strand by terminal deoxynucleotidyl transferase. After second strand synthesis using oligo-dT primer, amplification of dsDNA is performed. The feature of this method is introduction of suppressed PCR (2), which reduce by-product of PCR. This method is published from RIKEN, Japan.
1. Sasagawa, Y. et al. Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity. Genome Biol 14, R31, doi:10.1186/gb-2013-14-4-r31 (2013).
2. Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A. & Lukyanov, S. A. An improved PCR method for walking in uncloned genomic DNA. Nucleic acids research 23, 1087-1088 (1995).
After reverse-transcription with primer including T7 promoter and oligo-dT, poly-A is added to the 3′ end of the first strand by terminal deoxynucleotidyl transferase. After second strand synthesis using oligo-dT primer, amplification of dsDNA is performed with in vitro transcription by T7 polymerase (IVT). Cel-Seq can produce strand specific information. But the problem is the highly 3′-end skew bias of amplification.
Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell reports 2, 666-673, doi:10.1016/j.celrep.2012.08.003 (2012).
d) Tang’s single cell RNA-seq
After reverse-transcription with oligo-dT anchored primer, poly-A is added to the 3′ end of the first strand by terminal deoxynucleotidyl transferase. After second strand synthesis using oligo-dT primer, amplification of dsDNA is performed by PCR.
Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature methods 6, 377-382, doi:10.1038/nmeth.1315 (2009).
By reverse-transcription with oligo-dT anchored primer with reverse-transcriptase from the Moloney murine leukemia virus (M-MLV RT). As An oligo consisting of guanine residues and barcode are included in the reaction of RT, barcode sequence is added to 5′ end. After performing the biotinated single primer PCR, 5′ end of cDNAs are collected with Streptavidin beads leading to library construction.
Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21, 1160-1167, doi:10.1101/gr.110882.110 (2011).
f) Digital quantitative single-cell RNAs-seq
After reverse-transcription with oligo-dT anchored primer, assign unique ID to each product during or after synthesizing dsDNA. By this unique ID tagging, you can know the real number of reads even after PCR amplification.
1. Islam, S. et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nature methods 11, 163-166, doi:10.1038/nmeth.2772 (2014).
2. Shiroguchi, K., Jia, T. Z., Sims, P. A. & Xie, X. S. Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proceedings of the National Academy of Sciences of the United States of America 109, 1347-1352, doi:10.1073/pnas.1118018109 (2012).
3. Kivioja, T. et al. Counting absolute numbers of molecules using unique molecular identifiers. Nature methods 9, 72-74, doi:10.1038/nmeth.1778 (2012).
4. Casbon, J. A., Osborne, R. J., Brenner, S. & Lichtenstein, C. P. A method for counting PCR template molecules with application to next-generation sequencing. Nucleic acids research 39, e81, doi:10.1093/nar/gkr217 (2011).