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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. 
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RACE - Rapid Amplification of cDNA Ends02:35

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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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Ribosome Profiling02:24

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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.
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Complementary DNA01:44

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Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling
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Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

Published on: October 7, 2021

cDNA library generation from ribonucleoprotein particles.

Mathieu Rederstorff1, Alexander Hüttenhofer

  • 1Innsbruck Medical University, Innsbruck Biocenter, Division for Genomics and RNomics, Innsbruck, Austria. mathieu.rederstorff@maem.uhp-nancy.fr

Nature Protocols
|February 5, 2011
PubMed
Summary
This summary is machine-generated.

This study presents a new protocol to create cDNA libraries enriched for functional noncoding RNAs (ncRNAs). By isolating ncRNAs within ribonucleoprotein particles (RNPs), this method enhances the representation of biologically relevant ncRNA species for deep sequencing.

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

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

Published on: October 27, 2011

Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Noncoding RNAs (ncRNAs) are frequently bound by RNA-binding proteins, forming ribonucleoprotein particles (RNPs).
  • Standard methods for generating ncRNA cDNA libraries may underrepresent functional ncRNA species by excluding protein interactions.

Purpose of the Study:

  • To develop a protocol for generating specialized cDNA libraries highly enriched for functional ncRNA species.
  • To improve the representation of biologically relevant ncRNAs in deep sequencing libraries.

Main Methods:

  • Cellular extracts were fractionated using glycerol gradients to isolate RNPs.
  • ncRNAs were isolated from RNPs, followed by 3'-tailing and 5' adapter ligation.
  • Reverse transcription using an oligo-d(G) anchor primer generated cDNA, which was then deep-sequenced.

Main Results:

  • The RNP-based selection protocol significantly increased the proportion of functional ncRNA species in the cDNA library.
  • This method provides a higher probability of capturing biologically relevant ncRNAs compared to protein-devoid ncRNA library preparation.
  • Deep-sequencing-compatible cDNA libraries for functional ncRNAs can be generated within one week.

Conclusions:

  • The described protocol offers an efficient method for enriching functional ncRNAs in cDNA libraries through RNP isolation.
  • This approach is valuable for deep sequencing studies aiming to identify and characterize functional ncRNAs.
  • The protocol facilitates the generation of comprehensive ncRNA libraries for biological research.