Cap Analysis Of Gene Expression – Unlike microarrays and RNA-seq, CAGE can identify transcription start sites (TSS) and corresponding promoter regions by sequencing the 3′ end of cDNA (5′ end of RNA). This makes CAGE a powerful tool for analyzing gene regulation at the TSS level, allowing analysis of genes regulated by multiple alternative promoters. Therefore, CAGE can serve as a new perspective for genome annotation by elucidating transcriptional signaling cascades and performing other functions.
Gene expression gate analysis is a promoter detection and transcription analysis method developed by RIKEN (Patent No.: US 6174669, US 6221599, US8809518, etc.). CAGE uses a “cap-coating” technology based on biotinylation of the 7-methylguanosine cap of the Pol II transcript to extract reverse-transcribed 5 full-length cDNAs from the captured transcripts. Massively parallel sequencing and sequence tag analysis of cDNA 5′ ends reveals genome-wide transcription start sites and transcript extent. Therefore, CAGE provides efficient genome-wide transcriptional analysis as an alternative to microarrays and RNA-seq.
Cap Analysis Of Gene Expression
Map positions, read counts, CTSS clustering, differential expression analysis, gene ontology enrichment analysis, and transcription factor motif searches.
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First-strand cDNA synthesis is performed using our proprietary technology that facilitates thermostable reverse transcription and random priming. Random priming detects non-polyadenylated RNA. Removal of uncapped RNA, such as rRNA, truncated RNA, and fully reverse-transcribed RNA, by capping.
After selection of full-length cDNA, adapters are covalently bound to the 5′ and 3′ ends of the full-length enriched cDNA. CAGE tags are ready for high-throughput sequencing after 2-way synthesis.
CAGE tags were mapped to the reference genome to identify TSS and their promoter regions. Additionally, comparison of map positions with identified transcripts provides CAGE tag annotation. The number of CAGE tags generated at specific locations in the genome provides a genome-wide estimate of promoter activity.
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Therefore, CAGE can contribute to genome annotation, gene discovery, gene expression profiling, and promoter identification projects. The reference list provides examples of how CAGE can be successfully used in studies related to transcriptome analysis and promoter identification. Our CAGE protocol is based on a well-established procedure capable of acquiring large numbers of tags.
Dr. Oleg Gusev “CAGE allows us to see not only protein-coding genes, but also long non-coding RNAs and the recently discovered enhancer RNAs.”
PhD. Profiles developed by RIKEN (Shiraki, Kondo, et al., 2003; Kodzius, Kojima, et al., 2006). CAGE uses a “cap-coating” technology based on biotinylation of the 7-methylguanosine cap of the Pol II transcript to extract reverse-transcribed 5 full-length cDNAs from the captured transcripts. Massively parallel sequencing and sequence tag analysis of cDNA 5′ ends reveals genome-wide transcription start sites and transcript extent.
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For yeast, various techniques have been used to identify TSS genome-wide, such as microarrays (Hurowitz and Brown 2003, David, Huber et al. 2006, Xu, Wei et al. 2009), SAGE (gene sequence analysis) expression) (Zhang and Dietrich 2005), full-length cDNA clone sequencing (Miura, Kawaguchi et al. 2006), and RNA sequencing (Nagalakshmi, Wang et al. 2008). Microarray methods cannot provide single-nucleotide resolution of TSS due to limitations in probe design. It is well known that RNA-seq has the disadvantage of accurately identifying transcriptomes (Batout, Dobin et al., 2013; Steiger, Abril et al., 2013, Boley, Stoiber et al., 2013) because RNA-seq read sets are often expanded. transcribed into the final database (Wang, Gu et al. 2010, Graberr, Haas et al. 2011), which often lacks information on other TSS and their use. Sequencing of full-length cDNA clones probing the 5′ end of the RNA allows for more precise identification of TSS (Miura, Kawaguchi et al., 2006). However, the throughput is insufficient to provide sufficient information for quantitative measurement of lowly expressed genes and TSS usage (Shiraki, Kondo et al., 2003). Transcript isotope sequencing (TIF-seq) (Pelehano, Wei et al. 2014) also captures the 7-methylguanosine cap structure in transcript 5 and sequences the transcript using a high-throughput sequencer (e.g., Illumina) . TIF-seq is biased toward short RNA molecules, a common problem with this full-length cDNA sequencing method (Miura, Kawaguchi, et al., 2006). TIF-seq also requires PCR amplification and restriction enzyme digestion.
The TSS profiles we generated are based on the new CAGE protocol “nAnT-iCAGE” (Murata, Nishiori-Sueki, et al., 2014), which is unbiased as it does not involve PCR amplification, restriction enzyme digestion, or hybridization ( Murata)). , Sirai-Sueki et al. 2014).
TSSs in small genomic regions reflect the transcriptional activity of this core promoter. TSSs that were highly enriched within 20 bp of each other were clustered into tag clusters (TCs). If the boundaries of TCs (10% quantal position and 90% quantal position) are smaller than 50 bp, they are clustered together, corresponding to non-overlapping consensus clusters (core promoters).
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Currently, the Yeast TSS Map contains digital TSS maps and predicted core promoters from 12 yeast species, including 10 yeast species (e.g., Saccharomyces cerevisiae and the human pathogen Candida albicans) and two yeast species (including S. yeast). Pompeii). More types of data will be added when completed. For Saccharomyces cerevisiae, we used strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), which is a haploid derivative of laboratory strain S288c.
All strains were grown in YPD medium at 30°C to mid-log phase to isolate total RNA. In addition to YPD, S. cerevisiae was grown under eight other growth conditions that mimicked the natural environment (Table 1 ).
The location and activity of TSSs are valuable for precise identification of protein-coding genes and 5′ untranslated regions (5’UTRs), improving genome annotation, novel genes, core promoter elements, and transcription factors (TFs). Binding sites and other motifs associated with transcription and gene regulatory networks.
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Accessing the JBrowse genome browser takes users to a page where they can view TSS and core promoters with Gencode gene annotations. This guide will walk you through understanding trajectories in Genome Browser, as well as some tips for configuring JBrowse.
Move the view by clicking and dragging the track area, or clicking the navigation bar or the left and right arrow keys.
Center the view by clicking the track scale or overview line or moving within the track area.
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While holding down Shift, click the navigation bar or the up and down arrow keys to zoom in. Select an area and zoom in (“rubber band” zoom) by clicking the overview or track scale, or Shift-clicking and dragging in the track area.
Drag or double-click a track label from the Available Tracks area to the genome area. Turn off track labels by moving them from the genome region to the “Available Tracks” region.
Jump to a function or reference sequence by typing its name in the Location box and pressing Enter. Jump to a specific area by typing the area in the location box: ref:start..end.
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McMillan J, Lu Z, Rodriguez J, An TH, Lin Z. Database (Oxford) 2019.
Batout, P., A. Dobin, S. Plessy, P. Kaninch and T. R. Gingerglass (2013). “High-fidelity promoter analysis reveals extensive alternative promoter usage and transposon-driven developmental gene expression.” Genome Research 23(1):169-180.
Boley, N., M. H. Stoiber, B. V., Booth, K. H. Wang, R.A. Hoskins, P.J. Bickel, S. “Genome-guided transcriptome assembly through comprehensive analysis of RNA-seq data.” Nature Biotechnology 32(4):341-346.
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David, L., W. Huber, M. Granovskaya, J. Todlin, S. J. Palm, L. “High-resolution map of yeast genome transcription.” Continued Natl Acad Sci U S A 103(14):5320-5325.
Graeber, M. G., B. J. Haas, M. Yasur, J. Z. Levine, D.A. Thompson, I. Amit, . Regev (2011). “Assembling full-length transcriptomes from RNA-Seq data without a reference genome.” Nature Biotechnology 29(7):644-652.
Hurowitz, E.H. and P. profound. Brown (2003). “Genome-wide analysis of Saccharomyces cerevisiae mRNA length.” Genome Biology 5(1):R2.
Gene Expression Analysis
Kotzius, R., M. Kojima, H. Sairai, M. Nakamura, S. Fukuda, M. Tagami, D. Sasaki, K.). “CAGE: Gated analysis of gene expression.” Nature Methods 3(3): 211-222.
Miura, F., N. Kawaguchi, J. Sese, A. Toyoda, M. Hattori, S.
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