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Related Concept Videos

RNA-seq03:21

RNA-seq

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 microarray-based...
Ribosome Profiling02:24

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
RNA Splicing01:32

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...

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Related Experiment Video

Updated: May 11, 2026

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms
09:30

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Published on: September 13, 2018

Fragmentation of whole-transcriptome RNA using E. coli RNase III.

Manuel Ares

    Cold Spring Harbor Protocols
    |May 3, 2013
    PubMed
    Summary

    RNase III enzyme effectively fragments RNA for high-throughput sequencing (HTS) library preparation. This method generates RNA fragments with desired 5' phosphate and 3' OH ends, crucial for subsequent polymerase chain reaction (PCR) amplification and sequencing.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genomics

    Background:

    • High-throughput sequencing (HTS) enables massive parallel sequencing of RNA molecules for abundance measurement.
    • Quantifying RNA requires fragmentation into short, sequenceable pieces, ideally with 5' phosphate and 3' hydroxyl (OH) ends for efficient library construction.
    • Enzymatic RNA fragmentation that directly yields these desired ends simplifies library preparation by eliminating dephosphorylation or phosphorylation steps.

    Purpose of the Study:

    • To describe a standardized method for preparing fragmented transcriptome RNA for sequencing library construction.
    • To detail the use of RNase III for RNA fragmentation, focusing on generating fragments suitable for ligation and subsequent sequencing.
    • To outline the key steps involved in preparing RNA for sequencing libraries, including fragmentation, purification, and analysis.

    More Related Videos

    Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
    14:49

    Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

    Published on: October 27, 2011

    Related Experiment Videos

    Last Updated: May 11, 2026

    Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms
    09:30

    Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

    Published on: September 13, 2018

    Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
    14:49

    Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

    Published on: October 27, 2011

    Main Methods:

    • RNA fragmentation using RNase III to produce fragments of approximately 60-100 nucleotides.
    • Purification of fragmented RNA from the RNase III reaction mixture.
    • Analysis of the resulting RNA fragments to assess their suitability for sequencing library production.

    Main Results:

    • RNase III efficiently fragments RNA, yielding fragments with 5' phosphate and 3' OH termini.
    • The fragmentation process does not significantly alter the distribution of RNA sequencing reads, ensuring representative sampling.
    • The described three-step protocol (fragmentation, purification, analysis) is effective for preparing RNA for library construction.

    Conclusions:

    • RNase III is a valuable enzyme for RNA fragmentation in HTS library preparation due to its ability to generate desired end structures.
    • This method provides a streamlined approach to transcriptome sequencing library construction, enhancing efficiency and representation.
    • The described protocol offers a reliable standard for preparing fragmented RNA for downstream sequencing applications.