Quartz-Seq
Quartz-Seq
The Quartz-Seq method optimizes whole-transcript amplification (WTA) of single cells. in this method, a reverse-transcription (RT) primer with a T7 promoter and PCR target is first added to extracted mRNA. Reverse transcription synthesizes first-strand cDNA, after which the RT primer is digested by exonuclease I. A poly(A) tail is then added to the 3' ends of first-strand cDNA, along with a dT primer containing a PCR target. After second-strand generation, a blocking primer is added to ensure PCR enrichment in sufficient quantity for sequencing. Deep sequencing allows for accurate, high-resolution representation of the whole transcriptome of a single cell.
Pros:
- Single-tube reaction suitable for automation
- Digestion of RT primers by exonuclease I eliminates amplification of byproducts
- Short fragments and byproducts are suppressed during enrichment
Cons:
- PCR biases can underrepresent GC-rich templates
- Amplification errors caused by polymerases will be represented and sequenced incorrectly
- Targets smaller than 500 bp are preferentially amplified by polymerases during PCR