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Related Concept Videos

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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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Related Experiment Video

Updated: Oct 31, 2025

3' End Sequencing Library Preparation with A-seq2
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Application and design considerations for 3'-end sequencing using click-chemistry.

Madeline K Jensen1, Nathan D Elrod1, Hari Krishna Yalamanchili2

  • 1Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, TX, United States.

Methods in Enzymology
|June 29, 2021
PubMed
Summary

Researchers developed polyA-Click-seq (PAC-seq), a streamlined method using click chemistry for sequencing mRNA 3' ends. This technology aids in studying alternative polyadenylation (APA) and transcriptomic diversity in eukaryotic cells.

Keywords:
3′-end sequencingAlternative polyadenylationPAC-seqPolyA-miner

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Alternative polyadenylation (APA) is crucial for transcriptomic diversity.
  • Understanding APA's role in cellular physiology and pathology requires advanced detection technologies.
  • Previous methods for genome-wide APA analysis have limitations.

Purpose of the Study:

  • To present a streamlined protocol for polyA-Click-seq (PAC-seq) for mRNA 3'-end sequencing.
  • To provide a complete experimental and computational pipeline for analyzing mRNA 3'-end usage.
  • To enhance existing APA detection methods with improved library preparation.

Main Methods:

  • Utilized click-chemistry for efficient mRNA 3'-end library construction.
  • Incorporated spike-ins and unique molecular identifier primers for improved data accuracy.
  • Developed PAC-seq protocol for genome-wide monitoring of cleavage and polyadenylation events.

Main Results:

  • Demonstrated a streamlined protocol for PAC-seq library preparation.
  • Provided guidelines for optimizing sequencing depth and data analysis.
  • Integrated PAC-seq with the PolyA-miner computational tool for comprehensive analysis.

Conclusions:

  • PAC-seq offers an efficient method for analyzing mRNA 3'-end usage.
  • The combined PAC-seq and PolyA-miner pipeline provides a robust approach for studying APA.
  • This methodology advances the study of transcriptomic diversity and its implications in eukaryotic cells.