STRT
STRT
Single-cell tagged reverse transcription sequencing (STRT) is a method similar to CEL-seq that involves unique barcoding and sample pooling to overcome the challenges of samples with limited material. In this method, single cells are first picked in individual tubes, where first-strand cDNA synthesis occurs using an oligo(dT) primer with the addition of 3-6 cytosines. A helper oligo promotes template switching, which introduces the barcode on the cDNA. Barcoded cDNA is then amplified by single-primer PCR. Deep sequencing allows for accurate transcriptome sequencing of individual cells.
Pros:
- Barcoding and pooling allows for multiplexing and studying many different single cells at a time
- Sample handling and the potential for cross-contamination are greatly reduced due to using one tube per cell
Cons:
- PCR biases can underrepresent GC-rich templates
- Non-linear PCR amplification can lead to biases affecting reproducibility
- Amplification errors caused by polymerases will be represented and sequenced incorrectly
- Loss of accuracy due to PCR bias
- Targets smaller than 500 bp are preferentially amplified by polymerases during PCR