SCRB-Seq
Single-Cell RNA Barcoding and Sequencing
SCRB-Seq is a cost-efficient, multiplexed, single-cell mRNA sequencing technique (Soumillon et al., 2014). 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.
Advantages:
- Cost-efficient, high-throughput, single-cell transcriptome profiling
- Highly sensitive gene-detection results compared to popular scRNA-Seq techniques (Ziegenhain et al., 2016)
Disadvantages:
- Template-switching RT is heavily biased to full-length mRNA (Shapiro et al., 2013)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
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
References:
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