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A more efficient, sensitive & robust method of chromatin immunoprecipitation

A more efficient, sensitive & robust method of chromatin immunoprecipitation. Epigenetics: the study of the organisation and management of the genome. Proteins around which DNA is wound. Epigenetic landscape. Nucleosomes. CHROMOSOME. ME. Will affect health outcomes. Histone Tails.

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A more efficient, sensitive & robust method of chromatin immunoprecipitation

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  1. A more efficient, sensitive &robust method of chromatin immunoprecipitation

  2. Epigenetics: the study of the organisation and management of the genome Proteins around which DNA is wound Epigeneticlandscape Nucleosomes CHROMOSOME ME Will affect health outcomes Histone Tails Epigenetic marks are left by external factors such as: There are seven types of epigenetic mark in all including: Epigenetic marks • Environment • Ageing • Diet • Drugs • DNA methylation • Histone modifications • Nucleosome positioning • MicroRNA • Are reversible • Will affect the availability of genes to be activated • Will affect gene expression

  3. Example: Epigenetics in cancer research Epigeneticmarks CHROMOSOME ME Me Normal Normal • Disease development research examples: • HDAC inhibitors Me Me Me • Cyclical tetrapeptides • Preclinical to phase II • Short chain fatty acids • Phase I and II • Hydroxamic acids • Preclinical to Phase II Abnormal Abnormal Me Me Me Abnormal methylation of histone tails typically seen in cancer Changes in histone acetylation typically seen in cancer

  4. Effective chromatin immunoprecipitation (“ChIP”) is critical to epigenetic research Chip isolates specific epigenetic sites: CHROMOSOME 1. Chromatin sheared into fragments • Chip assays: • Complex • Difficult • Slow • Require skill/experience 2. Specific antibody added 3. Reverse cross link to release DNA from protein Therefore bottleneck In epigenetic research Proteinase K digestion: Selectively enriched DNA 4. PCR or ChIP SEQ to investigate site specific marks

  5. Targeting the Epigenetic landscape Complex, reversible context to gene expression and disease process regulation Seven epigenetic marks: Architecture proteins CTCF/Cohesin 5-hydroxy-methylctosine DNA methylation Histone variants P Me Remodeller CTCF Me Me DNA ncRNA Me Ac Histone modifications Remodelling complexes Non coding RNA (long non coding RNA) HDACi DNMTi HATi HMTi Multiple targets, from readers, writers and erasers to manipulate the chromatin landscape and reprogram disease gene expression profiles Enhanced understanding of aberrant disease mechanisms leads to targeted therapies (DNMT inhibitors, HDAC Inhibitors (Phase 1 trials)

  6. Chromatrap offers an inert solid-phase scaffold for better immunoprecipitation 1. Chromatin/antibody complex flows through matrix Protein A or G covalently bonded to matrix 2. Chromatin binds to protein 3. Everything else is flushed away 4. Chromatin released into fresh solution

  7. High surface area and flow-through characteristics are key to efficiency • Solid phase scaffold • Inert material • Open structure • Surface modified • Flow through process Promotes molecular movement Better sample mixing Minimises non specific binding

  8. A simpler and faster process with less manual handling Typical chip process in the lab 5 - 15 wash steps requiring manual pipette handling and incubation Requires re-suspension and bead handling by manual pipette handling Separate columns, further manual handling. Loss of DNA inevitable 10 hours total TRADITIONAL METHODS 2 - 4hours Chromatin preparation Shearing Antibody binding to protein Antibody/Protein bindingto chromatin Wash steps to remove nonspecific binding Elute to release chromatin Reversecross link DNA purification PCR orChip Seq 1 hour 3 simple wash- through steps. No incubation Simple centrifugation Not required 5 hours total CHROMATRAP

  9. More efficient, sensitive and robust assays • More efficient in the lab • A faster, simpler process • Less manual handling • Chromatrap 96 allows automation Better assay performance • Robust signal to noise • Wide dynamic range of sample size • Designed for 1ug sample sizes • High Sensitivity • Suitable for low abundant targets • Suitable for more challenging cell types

  10. Robust signal to noise compared to traditional methods Robust signal: noise ratios Excellent DNA enrichment Good replication Chemukhinet al., 2011; BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation. Analytical Biochemistry . 15;412(2):183-8

  11. Wide dynamic range: suitable for all sample sizes Signal to noise clear across dynamic range Full Chromatrap range: 50ng to 7500ng Optimum range shown here: 100ng to 2000ng • Binding shown for high abundant targets • RNA pol II onto GAPDH

  12. Ideal for low chromatin loadings: offers significant assay flexibility advantages Excellent signal strength from 1µg sample size: Allows lower volume inputs More assays per sample Good results from smaller biopsies 1µg sample: ChromatrapRECOMMENDS smaller input sample sizes of 0.5µg- 7.0µg

  13. Signal strength: clear differentiation across epigenetic landscape % Real Signal (Input) in K562

  14. Signal strength allows clear results from difficult or low abundant targets % Real Signal (Input) HepG2 Excellent correlation between H3K4me3 and H3k27me3 over positive and negative gene targets

  15. High sensitivity allows clear results from challenging cell types Peripheral blood mononuclear cells: CD14+ and lymphocytes Difficult to isolate samples from blood: Good signal:noise H3 onto GAPDH Pre-Spin Post-Spin Platelet + Plasma Diluted Blood PBMC Ficoll® + Gran. Ficoll® RBC

  16. High sensitivity allows clear results for multiple epigenetic targets and multiple gene loci using small samples Endometrial Differentiation Pipelle Biopsy 1mm sample Digestion: collagenase DNAase Mixed cell population. Stromal and epithelial compartments

  17. Chromatrap 96: high throughput with speed and sensitivity Example plate layout for 96 reactions: • Up to 96 reactions processed simultaneously, all in one day • Compatible with automated liquid handling • Allows, for the first time: • Multiple antibody and gene targets • Parallel investigation of widespread effects • Large, reliable data sets collected efficiently • Multiple cell types tested simultaneously • Multiple samples tested simultaneously

  18. Chromatrap 96 example: subset of data collected.4 targets; 4 cell types; 5 target gene lociShows experimental flexibility

  19. Wide dynamic range and sensitivity allows ChIP-seq At least 10 ng of DNA is needed for Illumina compatible library preparation. Chromatrap exceeds this even from samples of less than 1000ng: % Real Signal (Input) DNA pull-down range: 50ng 700ng

  20. Wide dynamic range makes Chromatrap suitable for FFPE samples FFPE rat uterine tissue Prepare high-quality chromatin Paraffin removal Protein – DNA- Complex extraction ChIP by enzymatic digestion FFPE: results Chromatrap % Real Signal (Input) Competitor

  21. Chromatrap team capabilities Based in the Institute of Life Sciences Swansea Design of bespoke high throughput assays Design of protocols for challenging cell types Antibody validation Protocol optimisation

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