CAGE tags tend to start with an extra guanine (G) that is not encoded in the genome, which is attributed to the template-free 5′-extension during the first-strand cDNA synthesis[2] or reverse-transcription of the cap itself.
[8] In nanoCAGE (Plessy et al., 2010),[9] the 5′ ends or RNAs were captured with the template-switching method instead of CAP Trapper, in order to analyze smaller starting amounts of total RNA.
The CAGEscan methodology (Plessy et al., 2010),[9] where the enzymatic tag cleavage is skipped, and the 5′ cDNAs sequenced paired-end, was introduced in the same article to connect novel promoters to known annotations.
With HeliScopeCAGE (Kanamori-Katayama et al., 2011),[10] the CAP-trapped CAGE protocol was changed to skip the enzymatic tag cleavage and sequence directly the capped 5′ ends on the HeliScope platform, without PCR amplification.
In 2018, Cvetesic et al.[16] increased the sensitivity of CAP-trapped CAGE by introducing selectively degradable carrier RNA (SLIC-CAGE, "Super-Low Input Carrier-CAGE").