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Advances in Genome Biology and Technology 2019 (AGBT)

February 27 - March 2

Visit the following poster presentations powered by Covaris Adaptive Focused Acoustics® (AFA®):

Presenter: Hamid Khoja, Ph.D., Principal Scientist

Title: Optimizing a Dual Fixation Protocol to Study Protein Complexes Binding to Chromatin in vivo

Description: Chromatin immunoprecipitation coupled with next-generation sequencing technology (ChIP-Seq) has enabled researchers to generate high-resolution maps of genomic binding sites for transcription factors and other members of the chromatin associated machinery. Many epigenetic factors that associate with chromatin do so as components of large multi-subunit complexes in which only a subset of the members directly bind to chromatin 1. For example, members the mammalian SWI/SNF chromatin remodeling complex, BAF, which promotes proper gene expression and chromatin dynamics, commonly associate with chromatin in assemblies of over ten subunits 2, 3. Here, we tested a Covaris-developed chromatin sample preparation protocol to effectively study the interactions of protein complexes associated with chromatin. An initial crosslinking cocktail (DMA, DSG, and EGS) was used prior to carrying out the Covaris truChIP® Chromatin sample preparation protocol for high-throughput sequencing.  

The use of ChIP-seq to map the genomic binding of specific members of such complexes often requires stabilization of the complex via protein-protein crosslinking. ChIP requires optimization of fixation conditions for each tissue or cell line, and for each protein complex being studied. Importantly, optimization is required for the subsequent shearing of the chromatin to obtain fragments within the distribution size required for sequencing, while simultaneously preserving the integrity of the target epitope. Relative to a standard single fixation method, our results indicate that the Covaris-developed protocol enhanced signal over background at known binding sites for members of the BAF complex including the ATPase subunit, BRG14. Taken together, the results indicate that the dual fixation method improves the detection of chromatin-associated machinery in ChIP assays. With optimization of fixation and shearing times, this protocol is suitable for use in other cells lines and conditions.

 

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Details

Start:
February 27
End:
March 2
Website:
https://www.agbt.org/the-general-meeting/

Venue

JW Marco Marriott Resort
400 S Collier Blvd
Marco Island, FL 34145 United States

Details

Start:
February 27
End:
March 2
Website:
https://www.agbt.org/the-general-meeting/

Venue

JW Marco Marriott Resort
400 S Collier Blvd
Marco Island, FL 34145 United States