Capped small RNA sequencing (csRNA-seq) for genome-wide mapping of active transcription start sites and enhancer RNAs from total RNA
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ABSTRACT:
PROVIDER: PRJNA1210985 | ENA |
REPOSITORIES: ENA
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Project description:Profiling ongoing transcription reveals the complete active transcriptome, including unstable enhancer RNAs, and provides a dynamic view of transcription dynamics, enabling the study of underlying gene regulatory mechanisms. Here, we describe a detailed protocol for capped- small RNA- sequencing (csRNA-seq), a method that uses total RNA to capture actively initiating or ‘nascent’ RNA polymerase II transcripts, including unstable RNAs such as enhancer RNAs, and their transcription start sites (TSSs). csRNA-seq offers several advantages: (1) the decoupling of sample collection and processing; (2) compatibility with diverse sample types, including fresh, frozen, and fixed tissues, as well as various eukaryotic organisms; and (3) scalability. Purified RNA is further non-infectious, can be isolated from inactivated samples—including clinical specimens or pathogenic samples—transported, and studied under standard laboratory conditions. This accessible protocol empowers researchers with minimal experience in nascent transcriptomics to study gene regulation and transcription dynamics.
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Project description:The spatial and temporal regulation of transcription initiation is pivotal for controlling gene expression. Here, we introduce capped-small RNA-seq (csRNA-seq), which uses total RNA as starting material to detect transcription start sites (TSS) of both stable and unstable RNAs at single-nucleotide resolution. csRNA-seq is highly sensitive to acute changes in transcription and identifies an order of magnitude more regulated transcripts than RNA-seq. Interrogating tissues from species across the eukaryotic kingdoms identified unstable transcripts resembling enhancer RNAs, pre-miRNAs, antisense transcripts and promoter upstream transcripts in multicellular animals, plants and fungi spanning 1.6 million years of evolution. Integration of epigenomic data from these organisms revealed that histone H3 trimethylation (H3K4me3) was largely confined to TSS of stable transcripts, while H3K27ac marked nucleosomes downstream of all active TSS, suggesting an ancient role for post-translational histone modifications in transcription. Our findings demonstrate that total RNA is sufficient to identify transcribed regulatory elements and capture the dynamics of initiated stable and unstable transcripts at single nucleotide resolution in eukaryotes.
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Project description:Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNAs) libraries including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or “nascent” RNA polymerase II transcription initiation in tissues and clinical samples. Finally, we present an accelerated and optimized protocol for small fragment sequencing with AVITI that aligns seamlessly with available sRNA library kits to meet this need.
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Project description:Cappable-seq was used to map transcription start sites globally in wild type Bacillus subtilis. Total RNA was isolated from cells grown in LB media until exponential phase. RNA corresponding to transcription start sites was capped with a 5' biotin tag, which was used for enrichment via a pull down with streptavidin beads. Enriched RNA was converted to cDNA and then subjected to illumina sequencing.
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Project description:To identify TF binding motifs with position-dependent functions associated with transcription initiation, we performed csRNA-seq to map initiating transcripts in Drosophila melanogaster embryos. We started with cell culturing and csRNA-seq for data acquisition. This was followed by a csRNA-seq bioinformatics analysis comparing TSS with more vs. less nascent transcription.
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Project description:The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Our data describes more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveals epitranscriptome-wide dynamics of NAD-RNAs with age.ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.
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Project description:BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5â²-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3â²â5â² degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells. Two cell lines were used. K562 cells were mock-irradiated (control) or UVC-irradiated at two different doses (25 and 100 J/m^2). HF1 cells were UVC-irradiated (20 J/m^2) in three independent experiments (nfUV4,nfUV3a, and nfUV3b). In one experiment, HF1 cells were also treated with TNF (10 ng/mL) 1 h prior to UV irradiation (tnfpreUV2, paired with nfUV4).
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