© Copyright Porvair Filtration Group Limited Commercial In Confidence © Copyright Porvair Filtration Group Limited Commercial In Confidence A more efficient, sensitive & robust method of chromatin immunoprecipitation

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

© Copyright Porvair Filtration Group Limited Commercial In Confidence Example: Epigenetics in cancer research Epigenetic marks CHROMOSOME Normal Abnormal Abnormal methylation of histone tails typically seen in cancer Me Normal Abnormal Changes in histone acetylation typically seen in cancer Disease development research examples: HDAC inhibitors Cyclical tetrapeptides o Preclinical to phase II Short chain fatty acids o Phase I and II Hydroxamic acids o Preclinical to Phase II Me ME

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

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

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

© Copyright Porvair Filtration Group Limited Commercial In Confidence High surface area and flow-through characteristics are key to efficiency Solid phase scaffold o Inert material o Open structure o Surface modified Flow through process Promotes molecular movement Better sample mixing Minimises non specific binding

© Copyright Porvair Filtration Group Limited Commercial In Confidence A simpler and faster process with less manual handling TRADITIONAL METHODS 10 hours total CHROMATRAP 5 hours total Typical chip process in the lab Chromatin preparation Shearing Antibody binding to protein Antibody/ Protein binding to chromatin Wash steps to remove non specific binding Elute to release chromatin Reverse cross link DNA purification PCR or Chip Seq 1 hour hours 3 simple wash- through steps. No incubation wash steps requiring manual pipette handling and incubation Simple centrifugation Requires re- suspension and bead handling by manual pipette handling Not required Separate columns, further manual handling. Loss of DNA inevitable

© Copyright Porvair Filtration Group Limited Commercial In Confidence 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 o Designed for 1ug sample sizes High Sensitivity o Suitable for low abundant targets o Suitable for more challenging cell types

© Copyright Porvair Filtration Group Limited Commercial In Confidence Robust signal to noise compared to traditional methods Robust signal: noise ratiosExcellent DNA enrichmentGood replication Chemukhin et al., 2011; BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation. Analytical Biochemistry. 15;412(2):183-8

© Copyright Porvair Filtration Group Limited Commercial In Confidence Wide dynamic range: suitable for all sample sizes Binding shown for high abundant targets RNA pol II onto GAPDH Optimum range shown here: 100ng to 2000ng Full Chromatrap range: 50ng to 7500ng Signal to noise clear across dynamic range

© Copyright Porvair Filtration Group Limited Commercial In Confidence 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: Chromatrap RECOMMENDS smaller input sample sizes of 0.5µg- 7.0 µg

© Copyright Porvair Filtration Group Limited Commercial In Confidence Signal strength: clear differentiation across epigenetic landscape % Real Signal (Input) in K562

© Copyright Porvair Filtration Group Limited Commercial In Confidence Signal strength allows clear results from difficult or low abundant targets Excellent correlation between H3K4me3 and H3k27me3 over positive and negative gene targets % Real Signal (Input) HepG2

© Copyright Porvair Filtration Group Limited Commercial In Confidence High sensitivity allows clear results from challenging cell types Peripheral blood mononuclear cells: CD14+ and lymphocytes Diluted Blood Ficoll® Platelet + Plasma PBMC Ficoll® + Gran. RBC Pre-SpinPost-Spin Difficult to isolate samples from blood:Good signal:noise H3 onto GAPDH

© Copyright Porvair Filtration Group Limited Commercial In Confidence High sensitivity allows clear results for multiple epigenetic targets and multiple gene loci using small samples Endometrial Differentiation Pipelle Biopsy 1mm sample Mixed cell population. Stromal and epithelial compartments Digestion: collagenase DNAase

© Copyright Porvair Filtration Group Limited Commercial In Confidence 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 o Parallel investigation of widespread effects Large, reliable data sets collected efficiently Multiple cell types tested simultaneously Multiple samples tested simultaneously

© Copyright Porvair Filtration Group Limited Commercial In Confidence Chromatrap 96 example: subset of data collected. 4 targets; 4 cell types; 5 target gene loci Shows experimental flexibility

© Copyright Porvair Filtration Group Limited Commercial In Confidence 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:50ng700ng

© Copyright Porvair Filtration Group Limited Commercial In Confidence Wide dynamic range makes Chromatrap suitable for FFPE samples Prepare high-quality chromatin Paraffin removal Protein – DNA- Complex extraction ChIP by enzymatic digestion % Real Signal (Input) Chromatrap Competitor FFPE: results FFPE rat uterine tissue

© Copyright Porvair Filtration Group Limited Commercial In Confidence 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