simplified the sequencing of CAGE libraries on Illumina sequencers by skipping second-strand synthesis directly loading single-strand cDNAs (Low Quantity Single Strand CAGE, "LQ-ssCAGE"). increased the sensitivity of CAP-trapped CAGE by introducing selectively degradable carrier RNA (SLIC-CAGE, "Super-Low Input Carrier-CAGE"). updated the nanoCAGE protocol to use the tagmentation method (based on Tn5 transposition) for multiplexing. published the nAnTi-CAGE protocol, where capped 5′ ends are sequenced on the Illumina platform with no PCR amplification and no tag cleavage. combined CAP trapper, template switching, and 5′-phosphate-dependent exonuclease digestion in RAMPAGE to maximize promoter specificity. to cleave tags with EcoP15I and sequence them on the Illumina-Solexa platform. In 2012, the standard CAGE protocol was updated by Takahashi et al. With HeliScopeCAGE (Kanamori-Katayama et al., 2011), 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.
The CAGEscan methodology (Plessy et al., 2010), 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. Longer tags were cleaved with the type III restriction enzyme EcoP15I and directly sequenced on the Solexa (then Illumina) platform without concatenation. In nanoCAGE (Plessy et al., 2010), 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. In 2008, barcode multiplexing was added to the DeepCAGE protocol (Maeda et al., 2008). In DeepCAGE (Valen et al., 2008), the tag concatemers were sequenced at a higher throughput on the 454 “ next-generation” sequencing platform. to better detect the non-polyadenylated RNAs. Random reverse-transcription primers were introduced in 2006 by Kodzius et al. The original CAGE method (Shiraki et al., 2003) was using CAP Trapper for capturing the 5′ ends, oligo-dT primers for synthesizing the cDNAs, the type IIs restriction enzyme MmeI for cleaving the tags, and the Sanger method for sequencing them. On the other hand, this addition of Gs was also utilised as a signal to filter more reliable TSS peaks. When not corrected, this can induce erroneous mapping of CAGE tags, for instance to nontranscribed pseudogenes. This knowledge in turn allows a researcher to investigate promoter structure necessary for gene expression.ĬAGE 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 or reverse-transcription of the cap itself.
THE FIVE KAGE SERIAL
Unlike a similar technique serial analysis of gene expression (SAGE) in which tags come from other parts of transcripts, CAGE is primarily used to locate exact transcription start sites in the genome. Using a reference genome, a researcher can usually determine, with some confidence, the original mRNA (and therefore which gene) the tag was extracted from. Copy numbers of CAGE tags provide a digital quantification of the RNA transcript abundances in biological samples. The reason I say its either way though is because we've never seen how Raditz would act when faced with a large group of adversaries and if he goes the area of effect route he wins, but if he doesn't he gives the team a good chance to get one good hax move in.The output of CAGE is a set of short nucleotide sequences (often called tags) with their observed counts. The kage have fodder to sacrifice in preparation of downing Raditz and the alliance have heavy hitters who can actually do something. Round 3 explanation: Bringing the two groups together solves their individual weaknesses. Only Oonoki's hax has a shot of taking Raditz down but as mentioned earlier in this thread, Raditz isn't fond of tanking and they don't survive long enough to coordinate an effective strategy. Round 2 Explanation: The only meaningful member here is Oonoki.Tsunade, A, and Mei have nothing to down Raditz and Gaara's sealing was easily busted out of by Madara. With that, a bunch of fodder have no chance of besting Raditz. Round 1 Explanation: I'm assuming Naruto and Sasuke aren't present as they're more powerful than the Kages and it'd be redundant to have the stronger group be round 1.