An optimized FAIRE procedure for low cell numbers in yeast.
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ABSTRACT:
PROVIDER: PRJNA411771 | ENA |
REPOSITORIES: ENA
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Project description:We report an optimised low-input FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements) procedure to assay chromatin accessibility from yeast colonies. Sensitivity, specificity and reproducibility of the scaled-down method are comparable to that of regular, higher input amounts, assayed over a 100-fold range. The method enables epigenetic analysis of chromatin structure to be assayed without the need for liquid culture, thus opening the possibility to study wild yeasts that form colonies, or those can be isolated in sufficient amounts in situ, from environmental samples.
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Project description:We report an optimized low-input FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements-sequencing) procedure to assay chromatin accessibility from limited amounts of yeast cells. We demonstrate that the method performs well on as little as 4 mg of cells scraped directly from a few colonies. Sensitivity, specificity and reproducibility of the scaled-down method are comparable with those of regular, higher input amounts, and allow the use of 100-fold fewer cells than existing procedures. The method enables epigenetic analysis of chromatin structure without the need for cell multiplication of exponentially growing cells in liquid culture, thus opening the possibility of studying colony cell subpopulations, or those that can be isolated directly from environmental samples.
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Project description:The compaction degree of chromatin is intimately related to its functionality and active cis-regulatory elements typically exist within open chromatin regions depleted in nucleosomes (Heintzman et al. 2007; Boyle et al. 2008). These domains can be identified using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) that allows for enrichment of nucleosome-depleted genomic regions when cross-linked chromatin is subjected to phenol-chloroform extraction (Nagy et al. 2003; Hogan et al. 2006) Here, chromatin structure was analyzed in MCF7 and LNCaP cells using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) (Nagy et al. 2003; Hogan et al. 2006) Keywords: FAIRE-chip
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Project description:Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we first sought to understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin for 15 minutes, 1 hour, 6 hours, and 24 hours. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and enables semi-quantitative estimates of relative gene expression in this yeast model.
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