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Capped small RNA sequencing (csRNA-seq) for genome-wide mapping of active transcription start sites and enhancer RNAs from total RNA

ABSTRACT: Capped small RNA sequencing (csRNA-seq) for genome-wide mapping of active transcription start sites and enhancer RNAs from total RNA

PROVIDER: PRJNA1210985 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32004964_1.fastq.gz	Fastqsanger.gz
	SRR32004964_2.fastq.gz	Fastqsanger.gz
	SRR32004965.fastq.gz	Fastqsanger.gz
	SRR32004966.fastq.gz	Fastqsanger.gz
	SRR32004967.fastq.gz	Fastqsanger.gz

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Capped small RNA sequencing (csRNA-seq) for genome-wide mapping of active transcription start sites and enhancer RNAs from total RNA

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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Sequencing short capped RNAs captures acute transcription initiation and identifies promoter and distal regulatory elements across eukaryotes from total RNA

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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