Schematic of the single-cell Quartz-Seq and Quartz-Chip methods. All of the steps of the whole-transcript amplification were executed in a single PCR tube. The first-strand cDNA was synthesized using the reverse transcription (RT) primer, which contains oligo-dT24, the T7 promoter (T7) and the PCR target region (M) sequences. After the first-strand synthesis, the majority of the RT primer was digested by exonuclease I, although it was not possible to eliminate the RT primer completely using this procedure. A poly-A tail was then added to the 3' ends of the first-strand cDNA and to any surviving RT primer. After the second-strand synthesis with the tagging primer, the resulting cDNA and the byproducts from the surviving primers contained the whole-transcript amplification (WTA) adaptor sequences, which include the RT primer sequence and the tagging primer sequence. These DNAs were used for the suppression PCR, which used the suppression PCR primer. Enrichment of the short DNA fragments, such as the byproducts, was suppressed. After the enrichment, the high-quality cDNA, which did not contain any byproducts, was obtained. The amplified cDNAs then had the T7 promoter sequence at the 3' ends of the DNA. These cDNAs were used for the Illumina sequencing and microarray experiments.
Sasagawa et al. Genome Biology 2013 14:R31 doi:10.1186/gb-2013-14-4-r31