Wellenreuther et al. (2004) utilized the SMART method to obtain large full-length clones.
Kapteyn et al. (2010) described using nucleotide isomers forming non-standard base pairs in template-switching oligonucleotides to prevent background noise during cDNA synthesis. In this approach, a modified TSO called iso3TS is to incorporate iso-C and iso-G bases at the 5' end of the oligonucleotide.
Salimullah et al. (2011) introduced the Cap Analysis Gene Expression (CAGE) method for identifying the 5’-ends in transcripts to discover new promotors and quantify gene activities.
Ramsköld et al. (2012) employed the TSO method for sequencing mRNAs from single cells and individual circulating tumor cells. This mRNA-sequencing protocol is called Smart-Seq. Smart-Seq utilizes the SMART™ template-switching technology to generate full-length cDNAs using only 12 to 18 cycles of PCR following the initial cDNA synthesis steps.
Shapiro et al. (2013) reviewed single-cell sequencing-based technologies for fundamental whole-organism analysis or studies.
Harbers et al. (2013) compared the performance of DNA-RNA, DNA-BNA(LNA), and DNA oligonucleotides in TSOs during nanoCAGE library preparation. Sequencing results revealed that DNA-RNA oligonucleotides showed the highest specificity for capped 5′-ends of mRNA, but the DNA-LNA provided a similar gene coverage with more reads falling within exons.
Picelli et al. (2014) published a protocol for Smart-seq2 allowing the generation of full-length cDNA and sequencing libraries using standard reagents. The entire protocol takes ∼two days, starting from cell picking to having a final library ready for sequencing, and sequencing requires an additional 1 to 3 days, depending on the strategy and sequencer used. Limitations of this protocol are the lack of strand specificity and the inability to detect non-polyadenylated (polyA(-)) RNA.
Turchinovich et al. (2014) utilized a similar ligation-independent method to generate DNA libraries for deep sequencing from picogram amounts of DNA and RNA.
Saliba et al. (2016) prepared single-cell libraries using a modified Smartseq2 protocol. The TSO utilized was altered by adding isomeric nucleotides at the 5’-end. The incorporation of these nucleotides minimizes background cDNA synthesis (TSO: 5'-(iso-dC)(iso-dG)(iso-dC)AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3'; +G = BNA/LNA).
Identifying genes' transcription start sites (TSS) is needed to characterize promotor regions. Cumbie et al. (2015) adapted a nano Cap Analysis of Gene Expression (nanoCAGE) method for the Illumina HiSeq-2000 sequencing platform. This method traps or captures the 5′-N7-Methylguanosine-triphosphate (7mG-p-p-p-N) modification common to all pol-II generated transcripts, known as the "cap," with streptavidin beads. The libraries were sequenced using the Illumina HiSeq-2000 platform with increased sequencing depth and excellent genome coverage.
A template-switching oligonucleotide (TSO) hybridizes to untemplated C nucleotides added by the reverse transcriptase during transcription. A TSO adds a 5’-sequence to full-length cDNA used for downstream cDNA amplification.
The reverse transcriptase switches templates and continues cDNA synthesis when encountering the cDNA-TSO cross-junction. Incorporating the 5′-adapter sequence into the TSO and using polyadenylation to prime reverse transcription avoids ligation steps.
Wulf et al. (2022) showed that chemical capping enhances template switching efficiency. The 5′-cap is a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA. This feature propels template switching. The research group introduced a sequencing library preparation workflow called CapTS-seq by combining chemical capping with template-switching reverse transcription as a new strategy for sequencing small RNAs. This method combines chemical capping to add a synthetic cap onto 5′-monophosphate RNAs with template switching. The study of various non-native synthetic cap structures revealed that an unmethylated guanosine triphosphate cap has the lowest bias and highest efficiency for template switching.
The CapTS-seq method allows synthetic miRNAs, human total brain and liver FFPE RNA sequencing and improves library quality for miRNAs.
Single-cell 3’-assays contain a polyd(T) sequence as part of the gel bead oligonucleotide and a barcode.
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