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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Mapping and quantifying nascent transcript start sites using TT-TSS-seq.

Eleanor Elgood Hunt1, Claudia Vivori1, Richard Mitter1

  • 1The Francis Crick Institute, London, NW1 1AT, United Kingdom.

Genome Research
|February 17, 2026
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Summary

We developed TT-TSS-seq to map transcription start sites from nascent RNA. This method accurately quantifies initiation sites, revealing insights into gene regulation and unstable transcripts.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Gene Regulation

Background:

  • Transcription initiation is a complex, regulated process determining gene expression.
  • Accurate mapping of transcription start sites (TSSs) from nascent RNA is crucial for understanding transcription dynamics.
  • Existing methods face challenges in sensitivity and accuracy, especially for unstable transcripts.

Purpose of the Study:

  • To develop and validate a sensitive method for mapping and quantifying transcription initiation sites from nascent RNA.
  • To improve the accuracy and sensitivity of transcription start site sequencing (TSS-seq) for low-input RNA samples.
  • To investigate transcription initiation patterns, including the use of multiple TSSs and the detection of unstable transcripts.

Main Methods:

  • Combined transient transcriptome sequencing with optimized transcription start site sequencing (TT-TSS-seq).
  • Refined enzymatic reactions for decapping and RNA ligation to enhance sensitivity.
  • Incorporated 5' oligonucleotides with unique molecular identifiers (UMIs) and barcodes for accurate quantification and multiplexing.

Main Results:

  • Successfully mapped and quantified transcription initiation sites from nascent transcripts using TT-TSS-seq.
  • Demonstrated the method's ability to detect transcription initiation of unstable transcripts, such as enhancer RNAs.
  • Revealed that a significant proportion of genes utilize multiple transcription initiation sites, often leading to a single stable transcript.

Conclusions:

  • TT-TSS-seq offers precise mapping and quantification of transcription initiation sites.
  • The method provides valuable insights into transcriptional dynamics and gene regulation.
  • TT-TSS-seq expands the available tools for studying gene expression at the initiation level.