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
Poster number : 520
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.
Presenter: Shurjo K. Sen, National Cancer Institute
Title: Hidden Comonents of MicroBiome Diversity Revealed by Cell Wall Lysis with Adaptive Focused Acoustics
Poster number : 718
Description: Within the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm, and some species are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly results in some species being missed altogether.
To develop a cell wall thickness-agnostic microbiome lysis protocol, we used Adaptive Focused Acoustics (AFA), a cutting-edge methodology for processing of biological samples. AFA employs focused bursts of ultrasonic acoustic energy at a very high frequency, producing a wavelength of only ~3mm and enabling energy to be focused into a discrete zone within a sample vessel. In a comparative 16S sequencing experiment with chemical/mechanical lysis, we show substantial increases in microbial alpha and beta diversity when stool pellets from the same mice are sequenced after AFA lysis. By starting with lower AFA levels, sequentially removing aliquots, and then subjecting these to progressively stronger AFA treatment, we developed a sequential lysis method that accounts for differences in cell wall thickness. Finally, we found that 46 OTUs were present only in AFA-lysed samples (i.e., completely missed by current chemical/mechanical lysis protocols), all of which were Gram positive. 16S results were verified by shotgun metagenome sequencing of a subset of the AFA samples.
We suggest that AFA produces a truer representation of the native microbiome, both in terms of reproducing species ratios and well as capturing increased diversity. We show that some bacterial species may escape undetected by current microbiome techniques.
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