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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: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE287021 | GEO | 2025/01/22

REPOSITORIES: GEO

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Project description:<p>Non-coding elements in our genomes that play critical roles in complex disease are frequently marked by highly unstable RNA species. Sequencing nascent RNAs attached to an actively transcribing RNA polymerase complex can identify unstable RNAs, including those templated from gene-distal enhancers (eRNAs). However, nascent RNA sequencing techniques remain challenging to apply in some cell lines and especially to intact tissues, limiting broad applications in fields such as cancer genomics and personalized medicine. Here we report the development of chromatin run-on and sequencing (ChRO-seq), a novel run-on technology that maps the location of RNA polymerase using virtually any frozen tissue sample, including samples with degraded RNA that are intractable to conventional RNA-seq. We used ChRO-seq to develop the first maps of nascent transcription in 23 human glioblastoma (GBM) brain tumors and patient derived xenografts. Remarkably, >90,000 distal enhancers discovered using the signature of eRNA biogenesis within primary GBMs closely resemble those found in the normal human brain, and diverge substantially from GBM cell models. Despite extensive overall similarity, 12% of enhancers in each GBM distinguish normal and malignant brain tissue. These enhancers drive regulatory programs similar to the developing nervous system and are enriched for transcription factor binding sites that specify a stem-like cell fate. These results demonstrate that GBMs largely retain the enhancer landscape associated with their tissue of origin, but selectively adopt regulatory programs that are responsible for driving stem-like cell properties. We also identified enhancers and their associated transcription factors that regulate genes characteristic of each known GBM subtype, and discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study uncovers new insights into the molecular etiology of GBM and introduces ChRO-seq which can now be used to map regulatory programs contributing to a variety of complex diseases.</p>

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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

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