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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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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...

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Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells
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Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

Sunil Kumar Raghav1, Bart Deplancke

  • 1Laboratory of Systems Biology and Genetics, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|September 23, 2011
PubMed
Summary

Multiplexed Chromatin immunoprecipitation sequencing (ChIP-Seq) enables simultaneous genome-wide profiling of multiple DNA-binding proteins. This cost-effective method enhances sensitivity and resolution for studying gene regulation.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Epigenetics

Background:

  • Chromatin immunoprecipitation (ChIP) identifies in vivo protein-DNA interactions.
  • ChIP coupled with microarray (ChIP-chip) or sequencing (ChIP-Seq) offers genome-wide DNA occupancy profiling.
  • ChIP-Seq surpasses ChIP-chip due to higher sensitivity, quantitation, and resolution, minimizing probe-related biases.

Purpose of the Study:

  • To provide a detailed protocol for performing multiplexed ChIP-Seq analyses.
  • To demonstrate the application of multiplexed ChIP-Seq for genome-wide RNA Polymerase II profiling.
  • To highlight the adaptability of the protocol for various DNA-binding proteins.

Main Methods:

  • Step-by-step guide for multiplexed ChIP-Seq.
  • Focus on genome-wide profiling of RNA Polymerase II.
  • Adaptable protocol for chromatin modifiers and transcription factors.

Main Results:

  • Multiplexed ChIP-Seq allows simultaneous analysis of multiple samples with sufficient sequence coverage.
  • This approach significantly reduces the cost per ChIP-Seq experiment.
  • Proof-of-concept demonstrates successful genome-wide profiling of RNA Polymerase II occupancy.

Conclusions:

  • Multiplexed ChIP-Seq is a sensitive, quantitative, and cost-effective method for genome-wide DNA-binding protein profiling.
  • The protocol facilitates the study of gene regulatory mechanisms by analyzing protein occupancy.
  • This technique is broadly applicable to various DNA-binding proteins, including transcription factors and chromatin modifiers.