SCRB-Seq
SCRB-Seq
SCRB-Seq is a cost-efficient, multiplexed, single-cell mRNA sequencing technique.
SCRB-Seq isolates single cells into wells using FACS. After cell lysis, poly(A)+ mRNAs are annealed to a custom primer containing a poly(T) tract, UMI, well barcode, and biotin. Template-switching RT and PCR amplification reactions are carried out on the mRNA, generating barcoded, full-length cDNA. cDNA strands from all wells are pooled together to be purified. They are PCR-amplified and purified further. The cDNA libraries are prepared using the Nextera XT library preparation protocol, with modified i5 primers. The resultant cDNA fragments are size-selected for 300–800 bp and sequenced.
Pros:
- Cost-efficient, high-throughput, single-cell transcriptome profiling
- Highly sensitive gene-detection results compared to popular scRNA-Seq techniques1
Cons:
- Template-switching RT is heavily biased to full-length mRNA2
- SCRB-Seq: Soumillon M., Cacchiarelli D., Semrau S., van Oudenaarden A. and Mikkelsen T. S. (2014). Characterization of directed differentiation by high-throughput single-cell RNA-Seq. bioRxiv
- 1. Ziegenhain C., Parekh S., Vieth B., Smets M., Leonhardt H., et al. (2016) Comparative analysis of single-cell RNA-sequencing methods. bioRxiv
- 2. Shapiro E., Biezuner T. and Linnarsson S. (2013) Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet 14: 618-630
- Ziegenhain C., Parekh S., Vieth B., Smets M., Leonhardt H., et al. Comparative analysis of single-cell RNA-sequencing methods. bioRxiv. 2016;
- Shapiro E., Biezuner T. and Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013;14:618-630
- Cacchiarelli D., Trapnell C., Ziller M. J., et al. Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency. Cell. 2015;162:412-424
- Anahtar M. N., Byrne E. H., Doherty K. E., et al. Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity. 2015;42:965-976