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<p>High-resolution mapping of active RNA polymerase II transcription initiation provides a dynamic view of gene expression and reveals the entire spectrum of RNA transcripts—from stable mRNAs to transient enhancer RNAs—which is critical for understanding gene regulation, deciphering transcriptional programs and defining regulatory element function. Here we present a detailed protocol for capped small RNA sequencing (csRNA-seq). Starting with total RNA, which can be readily isolated from fresh, frozen or fixed cells, tissues or patient samples, csRNA-seq selectively enriches for actively initiating 5′-capped RNA polymerase II transcripts. This approach captures both initiating stable protein-coding RNAs and non-coding RNAs, as well as rapidly degraded, transient transcripts such as enhancer or promoter divergent RNAs, providing a comprehensive snapshot of active <i>cis</i>-regulatory elements and facilitating the study of underlying regulatory mechanisms with high sensitivity. The protocol involves small RNA isolation, 5′-capped RNA enrichment and library generation, followed by sequencing. Key advantages of csRNA-seq over other nascent RNA-seq methods include (i) decoupling of sample collection and processing, (ii) broad compatibility with diverse eukaryotic sample types and organisms, (iii) high-resolution data defining active regulatory elements and their properties and (iv) scalability. Importantly, purified RNA is non-infectious and can be isolated from inactivated samples, including clinical or pathogenic specimens, allowing safe transport and analysis under standard laboratory conditions. This protocol empowers researchers with minimal experience in nascent transcriptomics to study gene regulation, <i>cis</i>-regulatory elements and transcription dynamics.</p>

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Profiling active RNA polymerase II transcription start sites from total RNA by capped small RNA sequencing (csRNA-seq)

  • 期刊论文

  • 发表日期：

    2026年1月16日

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  • Mackenzie K. Meyer,
  • Oluwadamilola J. Olanrewaju,
  • Patricia Montilla-Perez,
  • Anna L. McDonald,
  • Eva M. Rickard,
  • Francesca Telese,
  • Christopher Benner,
  • Marina I. Savenkova,
  • Sascha H. Duttke

摘要

High-resolution mapping of active RNA polymerase II transcription initiation provides a dynamic view of gene expression and reveals the entire spectrum of RNA transcripts—from stable mRNAs to transient enhancer RNAs—which is critical for understanding gene regulation, deciphering transcriptional programs and defining regulatory element function. Here we present a detailed protocol for capped small RNA sequencing (csRNA-seq). Starting with total RNA, which can be readily isolated from fresh, frozen or fixed cells, tissues or patient samples, csRNA-seq selectively enriches for actively initiating 5′-capped RNA polymerase II transcripts. This approach captures both initiating stable protein-coding RNAs and non-coding RNAs, as well as rapidly degraded, transient transcripts such as enhancer or promoter divergent RNAs, providing a comprehensive snapshot of active cis-regulatory elements and facilitating the study of underlying regulatory mechanisms with high sensitivity. The protocol involves small RNA isolation, 5′-capped RNA enrichment and library generation, followed by sequencing. Key advantages of csRNA-seq over other nascent RNA-seq methods include (i) decoupling of sample collection and processing, (ii) broad compatibility with diverse eukaryotic sample types and organisms, (iii) high-resolution data defining active regulatory elements and their properties and (iv) scalability. Importantly, purified RNA is non-infectious and can be isolated from inactivated samples, including clinical or pathogenic specimens, allowing safe transport and analysis under standard laboratory conditions. This protocol empowers researchers with minimal experience in nascent transcriptomics to study gene regulation, cis-regulatory elements and transcription dynamics.