Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.
View Article and Find Full Text PDFIn eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs.
View Article and Find Full Text PDFNon-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments.
View Article and Find Full Text PDFUsing the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs).
View Article and Find Full Text PDFCap analysis of gene expression (CAGE) is an approach to identify and monitor the activity (transcription initiation frequency) of transcription start sites (TSSs) at single base-pair resolution across the genome. It has been effectively used to identify active promoter and enhancer regions in cancer cells, with potential utility to identify key factors to immunotherapy. Here, we overview a series of CAGE protocols and describe detailed experimental steps of the latest protocol based on the Illumina sequencing platform; both experimental steps (see Subheadings 3.
View Article and Find Full Text PDFAlthough it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses.
View Article and Find Full Text PDFCap analysis of gene expression (CAGE) provides accurate high-throughput measurement of RNA expression. By the large-scale analysis of 5' end of transcripts using CAGE method, it enables not only determination of the transcription start site but also prediction of promoter region. Here we provide a protocol for the construction of no-amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE).
View Article and Find Full Text PDFCAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5' end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries.
View Article and Find Full Text PDFRegulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles.
View Article and Find Full Text PDFCap-analysis gene expression (CAGE) provides accurate high-throughput measurement of RNA expression. CAGE allows mapping of all the initiation sites of both capped coding and noncoding RNAs. In addition, transcriptional start sites within promoters are characterized at single-nucleotide resolution.
View Article and Find Full Text PDFWe provide here a protocol for the preparation of cap-analysis gene expression (CAGE) libraries, which allows for measuring the expression of eukaryotic capped RNAs and simultaneously map the promoter regions. The presented protocol simplifies the previously published ones and moreover produces tags that are 27 nucleotides long, which facilitates mapping to the genome. The protocol takes less than 5 days to complete and presents a notable improvement compared to previously published versions.
View Article and Find Full Text PDFWe developed a method for selective purification of DNA using the cationic detergent, cetyltrimethylammonium bromide (CTAB), accompanied with urea and controlled high-salt (NaCl) concentration. This method is effective for rapid separation of DNA fragments from artifacts such as PCR primer dimers or ligation adapters. The CTAB-associated purification completely removed the short PCR artifacts and primers, as well as enzymes and buffer, while recovering a sufficient quantity of amplicons for subsequent experiments such as preparation of libraries.
View Article and Find Full Text PDFIt has been reported that relatively short RNAs of heterogeneous sizes are derived from sequences near the promoters of eukaryotic genes. In conjunction with the FANTOM4 project, we have identified tiny RNAs with a modal length of 18 nt that map within -60 to +120 nt of transcription start sites (TSSs) in human, chicken and Drosophila. These transcription initiation RNAs (tiRNAs) are derived from sequences on the same strand as the TSS and are preferentially associated with G+C-rich promoters.
View Article and Find Full Text PDFUsing deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network.
View Article and Find Full Text PDFCAGE (cap analysis of gene expression) is a method for identifying transcription start sites by sequencing the first 20 or 21 nucleotides from the 5' end of capped transcripts, allowing genome-wide promoter analyses to be performed. The potential of the CAGE as a form of expression profiling was limited previously by sequencing technology and the labor-intensive protocol. Here we describe an improved CAGE method for use with a next generation sequencer.
View Article and Find Full Text PDFRice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function.
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