Training Solutions from Covaris for epigenomic applications Sean

Training: Solutions from Covaris for epigenomic applications Sean Gerrin, ALM Product Manager, Epigenomics www. covaris. com

Agenda • • • Introduction & product vision Brief overview of epigenomics Why customers study epigenomics and its significance Market size potential Product strategy Workflows and value propositions New products Tissue applications Conducting demos Demo success story Lead generation Discussion Proprietary 2

Looking beyond today and expanding outside of naked DNA shearing Focus on developing and leveraging core competencies of Covaris Vision Corporate Strategy Divisional Strategy Product/market strategy Tactical efforts Customer Satisfaction Reference: Gorchels, L. The Product Manager’s Handbook. New York: Mc. Graw Hill, 2006 Focus on customer specific needs Proprietary 3

Why do Covaris customer study epigenetics? Reference: Huang C, Wu JC. Epigenetic Modulations of Induced Pluripotent Stem Cells: Novel Therapies and Disease Models. Drug discovery today Disease models. 2012; 9(4): e 153 -e 160. doi: 10. 1016/j. ddmod. 2012. 004. Proprietary 4

A field that is growing rapidly Reference: https: //www. assistingnature. gr/blog/? p=544 Reference: Personalized Medicine, the Path Forward. Mc. Kinsey and Co. 2013. Proprietary 5

A growing segment within the NGS market Forecasted Market Trends • • NGS Market estimated to reach $8. 7 B by 2020 and 12. 45 B by 2022 CAGR of 20. 5% in the forecast period (2017 -2022) The global epigenetics market accounted to USD $757. 3 M in 2016 growing at a CAGR of 13. 0% during the forecast period of 2017 to 2024 The Ch. IP-Seq market is expected to grow at the highest CAGR during the forecast period (2017 -2022) Driving Factors • • Quality of the readout depends on the quality of the pre-sequencing treatment Demand for standardized sample preparation solutions Segmented by Application • • • Diagnostics Projected to account for the major share of the global NGS market Drug Discovery Biomarker Discovery Precision Medicine Segmented by End-User • • • Research Centers (academic & government) Hospitals & Clinics – Expected to register the highest CAGR Pharmaceutical & biotechnology companies References 1. http: //www. marketsandmarkets. com/Market-Reports/next-generation-sequencing-ngs-technologies-market-546. html? gclid=CLjy 97 z. N-NQCFQ 1 WDQod 8 Lc. Ofg 2. http: //www. marketwatch. com/story/next-generation-sequencing-ngs-market-growing-at-a-cagr-of-205 -during-2017 -to-2022 ---reportsnreports-2017 -04 -07 -1320318 3. http: //www. prnewswire. com/news-releases/ngs-market-growth-at-20 -cagr-to-2020 -next-gen-sequencing-industry-global-forecasts-in-new-research-report-274345491. html 4. https: //www. thestreet. com/story/14080978/1/next-generation-sequencing-ngs-market-by-product-hiseq-miseq-hiseq-x-tenx-five-nextseq 500 -ion-proton-pgm-ions 5 -pacbio-rsii-services-targeted-rna-exome-de-novo-application-diagnostics-biomarkeragriculture 5. http: //www. digitaljournal. com/pr/3658212 Proprietary 6

Strategy to grow Covaris in the epigenomics space Identify and profile customers Develop customer value profile Reach beyond existing demand: Attract new customers with similar customer profiles • • • Maintain relationship with customers Market Segments Pharma/biotech Clinical/diagnostic screening Academic research Customer Segments Low-throughput Mid throughput (neuroscience, oncology, immunology) High throughput (neuroscience, oncology, immunology) Support customers and keep labs current on new apps Help the customer scale workflows Proprietary 7

Overview of Ch. IP Sample Preparation Reference: https: //tools. thermofisher. com/content/sfs/brochures/Step-by-Step-Guide-to-Successful-Ch. IP-Assays. pdf Proprietary 8

tru. Ch. IP Sample Preparation Workflow for Cultured Cells Genetic Predisposition Fix cells with Formaldehyde • Determine the number of cells • Fix with 1% methanol-free formaldehyde Akt 1 Fkbp 5 • Quench with Glycine Isolate nuclei Nr 3 c 2 • Lyse cell membrane to release chromatin • Dissolve cell membrane and organelles Maoa • Release protein-DNA complexes Environmental Stress Nr 3 c 1 Childhood trauma Nutrition Drug abuse Maternal separation Bdnf Shear Chromatin IP Purification • Shear chromatin to appropriate size for sequencing using a focused. Social ultrasonicator stress • For Ch. IP-Seq, 150 -700 bp is ideal for Illumina platforms Drd 2 • Utilize a well-characterized Ch. IP-grade antibody • Select IP method (e. g. , magnetic beads) • Reverse crosslinks • PK digestion / RNase treatment • Purify DNA and prepare sample for HT-sequencing Proprietary 9

Positioning the tru. Ch. IP product line Positioning statement–how a product is to be perceived in minds of the customer, relative to the competition Why should I buy Covaris products for my epigenetic applications? What value does Covaris add to the customer’s workflows? The statement should identify the relevant market segment, frame of reference, product’s point of differentiation, and competitive edge To investigators, tru. Ch. IP is the brand of robust sample preparation for Ch. IPSeq that preserves protein-DNA interactions due to the gentle acoustic shearing used, thus providing a more accurate representation of the true in vivo biology. Proprietary 10

tru. Ch. IP – Product Concept and Purpose Easier and faster optimization (< 1 week for most cell lines) Optimized fixation strategy Reagents provided with tru. Ch. IP work optimally with acoustic shearing Lower IP failure rates (protein-DNA interactions preserved during shearing) Lower detergent in shearing buffer (0. 1%) Shearing buffer can be converted to IP buffer without having to dilute the sample significantly, thereby allowing >chromatin available for IP • Size distribution is within the range required for sequencing ensuring a more accurate representation of the true biology • Conversation starts at, “What is tru. Ch. IP? ” and has the potential to end at, “How can I start using it? ” • • • Proprietary 11

How tru. Ch. IP improves the reputation of the Covaris Brand Commonly reported issues by customers not using tru. Ch. IP • “I fix the cells directly in the media and the shearing results look very poor” • “I fix the cells at 37°C, and not sure why I see a significant amount of chromatin above 1 Kb; can you explain? ” • “I am shearing my chromatin in RIPA buffer and not getting good results; why? ” How does tru. Ch. IP improve the customer’s experience with Covaris products? • Lower sample failure rates • Improved results • Willing to recommend product to colleagues (future instrument and reagent sales) Proprietary 12

Key Value Propositions for Covaris Chromatin Shearing • Reproducible mechanical shearing technology to help improve the quality of the results • Gentle shearing (lower energy requirements) to ensure epitope integrity is maintained • Multiple platforms available to support all labs Bioruptor Versus Covaris AFA 2 X 107 Raw 264. 7 monocytes Proprietary 13

Thermal control: Maintain protein-DNA interactions Covaris AFA • Covaris micro. TUBE • 2 MPa; at 180 s Bath Sonicator Probe Sonicator • Microfuge tube • 2 MPa; at 600 s • Microfuge tube • 2 MPa; at 300 s air water Isothermal: Sample, Bath and Vessel • • • No thermal control AFA processing is isothermal as compared to bath and probe sonicators Controlled process allows for more rapid processing No thermal damage to samples Proprietary 14

Covaris Tubes: Engineered for isothermal processing • Shearing DNA/chromatin in PCR tubes/plates will introduce thermal biased shearing • Plastic PCR tubes absorb acoustic energy and transfer it as heat to sample • Plastic then acts as insulator preventing heat dissipation Proprietary 15

Example of Consumables available micro. TUBE-130 Snap Cap 6 X 16 mm milli. TUBE-1 m. L with AFA fiber Proprietary 16

Sample/instrument compatibility table Instrument Cell Input Maximum for micro. TUBE-130 Throughput for micro. TUBE Cell Input Maximum for milli. TUBE-1 m. L Throughput for milli. TUBE Tissue Mass M 220 3 X 106 1 3 X 107 1 20 -80 mg ME 220 3 X 106 1 -8 3 X 107 1 -4 20 -80 mg S 220 3 X 106 1 3 X 107 1 20 -120 mg 1 -4 20 -120 mg 1 -24 20 -120 mg E 220 Evolution 3 X 106 1 -8 E 220 3 X 106 1 -96 LE 220 3 X 106 1 -96 3 X 107 3 X 106 3 X 107 Note: For low input Ch. IP, the user should test 30 -50 k cells per time point for the optimization experiments. Once optimized, one can shear with inputs as low as 5, 000 without making modifications to the shearing conditions. Proprietary 17

Sample/instrument compatibility table M 220 ME 220 S 220 Evolution Mammalian Cells/tissue Yeast Plant C. Elegans Drosophila – Tissue/Embryos Drosophila - Cultured Insect Cells Sample Type E 220 LE 220 Proprietary 18

Ch. IP is expensive: Multiplexing 4 Ch. IP libraries with 75 M reads/sample Step Cost 4 Ch. IP libraries Covaris (3 M cells/sample processed in the micro. TUBE 130) $8. 67/reaction (<1% of the total cost) $34. 68 Antibody (Cell Signaling Technologies, m. Ab #9733, H 3 K 27 me 3) $30/reaction $120. 00 Dynabeads (Thermo Fisher Scientific) $5/reaction $20. 00 Library Prep $250 $1, 000. 00 Sequencing (Illumina, 1 X 50 SE $1, 600/lane Hi. Seq V 4 Chemistry) $1, 600. 00 q. PCR $25/sample $100. 00 Bio. Analyzer $15/sample $60. 00 Total Cost $2, 934. 68 Proprietary 19

More investigators trust Covaris for Ch. IP-Seq Sample Preparation Covaris Cited in the Literature for Ch. IP Applications 1200 987 Number of Publications 1000 800 550 600 400 200 0 16 41 73 2010 2011 2012 200 138 2013 Year 2014 237 2015 2016 2017 Proprietary 20

Product Information Part Number Item Name 520256 tru. Ch. IP Ultra-Low Chromatin with Formaldehyde (50) tru. Ch. IP Chromatin Shearing Kit with Formaldehyde (50) tru. Ch. IP Chromatin Shearing Tissue Kit with Formaldehyde (6) tru. Ch. IP native Chromatin Shearing Kit (25) 520257 tru. Ch. IP FFPE Chromatin Shearing Kit (25) 520156 520154 520237 Description Includes reagents required to run ultra-low inputs (<100 k). Includes reagents required to process >100 K to 30 M cells. Includes reagents and accessories required to process low mass inputs (20 mg) and high mass inputs (120 mg). Includes reagents required to process <100 K cells and up to 30 M cells for native chromatin sample prep. Includes reagents required to emulsify paraffin, rehydrate tissue, and shear soluble chromatin ready for IP. Proprietary 21

NEW: tru. Ch. IP FFPE Chromatin Shearing Kit Key takeaways • An active-based paraffin removal workflow for isolating soluble chromatin from archived FFPE tissues • 3 X faster than other FFPE Ch. IP workflows • The only high-throughput FFPE Ch. IP workflow Proprietary 22

NEW: tru. Ch. IP Native Chromatin Shearing Kit Lyse cells and shear chromatin Dilute chromatin with IP Dilution Buffer IP PK Digest/Reverse Purify DNA Library Prep Key Takeaways • Gentle shearing settings to effectively fragment chromatin without dissociating the protein-DNA interactions • Alternative to micrococcal nuclease (MNase) for histonebased Ch. IP • Allows AFA to be used for multiple chromatin applications Proprietary 23

AFA is a multi-sample and multi-application platform • AFA is established as the standard g. DNA shearing method for DNA methylation analysis in the Agilent Sure. Select protocol • AFA is also the shearing method used in the Nu. GEN Ovation Methyl-Seq protocol for profiling 5 m. C and the ox. BS-Seq protocol for 5 hm. C profiling applications Proprietary 24

DNA Methylation Analysis using Covaris AFA Workflow Highlights • The only high-throughput (up to 96 samples) sample preparation workflow available • Fully validated workflows to process 500 ng to 3 µg of g. DNA • Robust shearing for accurate methylation (Cp. G) profiling • Part of the Nu. GEN Ovation Methyl-Seq and Agilent Sure. Select protocols Proprietary 25

Key takeaway: Multiple applications supported by AFA Ch. IRP-Seq FAIRE-Seq Hi-C Adaptive Focused Acoustics (AFA) Ch. IP-Seq WGBS Methyl. Seq Proprietary 26

Benchtop Illumina platforms (DNA-Protein Interaction Analysis) Proprietary 27

Production Scale Illumina Sequencers (DNA-Protein Interaction Analysis) Proprietary 28

Tissue Ch. IP with the Covaris cryo. PREP Product Line CP 01 • • The ideal product for low throughput tissue prep 15 mg to 2 g of tissue A low-cost alternative to the CP 02 (~$1, 000 USD) Evaluate the function of the CP product and then upgrade to CP 02 Ideal for academic research labs DNA, RNA, protein (e. g. membrane) chromatin extraction applications No sample-to-sample contamination (compared to mortar and pestle) Simplifies cell lysis compared with dounce homogenizer • • • Easier operation compared to CP 01 (automated versus manual) 15 mg to 2 g of tissue Validated for use with the tru. Ch. IP Chromatin Shearing Tissue Kit DNA, RNA, protein (e. g. membrane) chromatin extraction applications No sample-to-sample contamination (compared to mortar and pestle) Efficient cell lysis compared with dounce homogenizer CP 02 Proprietary 29

Covaris Ch. IP Sample Preparation Workflow for Tissue Genetic Predisposition Fix tissue with Formaldehyde Cryofracture tissue with cryo. PREP® • Estimate tissue mass • Section Tissue into small pieces • Fix with 1% methanol-free formaldehyde Fkbp 5 Akt 1 Shear Chromatin IP Purification Childhood trauma Nutrition • After quenching, flash freeze tissue in liquid nitrogen in Covaris tissue. TUBE Nr 3 c 1 Drug abuse • Disrupt the tissue matrix with cryo. PREP to homogenize tissue Nr 3 c 2 • Transfer pulverized tissue to glass tube for the nuclei prep Maoa Isolate nuclei Environmental Stress • Lyse cell membrane to release chromatin • Dissolve cell membrane and organelles • Release protein-DNA complexes Maternal separation Bdnf Social stress Drd 2 • Shear chromatin to appropriate size for sequencing using a focused-ultrasonicator • For Ch. IP-Seq, 150 -700 bp is ideal for Illumina platforms • Utilize a well-characterized Ch. IP-grade antibody • Select IP method (e. g. , magnetic beads) • Reverse crosslinks • PK digestion / RNase treatment • Purify DNA and prepare sample for HT-sequencing Proprietary 30

Clinically-focused Ch. IP-Seq Publication from Nature • • Studied the effects the MYBMIM inhibitor that interferes with the assembly of MYB: CBP/P 300 complex Inhibitor was studied to reduce MYB occupancy at target genes responsible for aberrant leukemia Found that a significant reduction in MYB-containing H 3 K 2327 Ac sites when treated with MYBMIM compared to TG 3 control Used Covaris S 220 Focused-ultrasonicator Proprietary 31

cryo. PREP for preparing mouse brain tissues for Ch. IP-Seq • • • Determined regulatory binding of NPAS 1/NPAS 3 by Ch. IP-Seq in WT and mutant hippocampus RNA-Seq revealed changes due to loss of NPAS 1/3 Used Ch. IP-Seq to infect which changes were due to direct transcriptional regulation via binding of transcription factors Used 14 -15 mg of murine hippocampal brain tissue Processed using tru. Ch. IP Chromatin Shearing Tissue Kit and Covaris E 220 Proprietary 32

Key takeaways • AFA focused-ultrasonicators are more expensive, however, they provide more reproducible shearing and maintain protein-DNA interactions • If protein-DNA interactions are disrupted during shearing, IP will be unsuccessful • Protocols do not need to be optimized multiple times because of the consistency provided by AFA (one time optimization for a cell line is sufficient) • If shearing is poor (150 -700 bp), clustering efficiency will be significantly lower resulting in poor sequencing data • Downstream costs associated with library prep and sequencing are significant (>$1 K). Therefore, a reproducible sample preparation solution is imperative Solution: tru. Ch. IP from Covaris. Fixation, nuclei prep, and shearing have been thoroughly optimized on all Covaris platforms to work with all mammalian cell types • Bath sonicators energize the entire water bath to initiate cavitation inside the sample. The energy is unfocused leading to much less control over the shearing • Transducer degradation is an issue often reported about the Bioruptor with multiple service calls required that are expensive and result in instrument down time leading to project delays Proprietary 33

Demo Qualification Process Customer qualification by Sales Manager 1 Pre-demo conference call/meeting( held at least 2 weeks prior to the demo) 2 Information gathering Setting customer expectations Setting our expectations for the demo 3 Discussion/shipping of all the necessary instruments, kits and consumables 4 Carrying out the demo 5 Data analysis/Feedback Proprietary 34

Pre-demo preparation work • Review demo guide • Gather information about the planned demo (technical/commercial qualification) • Ensures you bring the correct instrument/consumables/racks/kit(s) • • • Define objectives Set expectations Timeline Post-demo analysis Results Proprietary 35

Sections of the Ch. IP demo form Proprietary 36

Demo Tips 1. Deliver the tru. Ch. IP protocol prior to the call/meeting for the investigator to review 2. Ensure the investigator understands what she/he needs to prepare ahead of time for the demo (*tru. Ch. IP works best with cells cultured as a suspension) 3. Ensure the investigator uses all buffers/reagents provided in the tru. Ch. IP Chromatin Shearing Kit and follows all steps including fixation (*tru. Ch. IP is only to be used with mammalian cells) 4. Covaris recommends for only mammalian cell Ch. IP demos to be performed 5. Ensure to provide the correct sample tubes, holders/racks/kit depending on the protocol planned for the demo 6. Investigator may run current protocol in parallel for a true side-by-side comparison 7. Fixation Tip: For stem and primary cells, Covaris recommends 2. 5 and 5 minutes. For all other cell types, we recommend 5 and 10 minutes. 8. Ensure crystals in quenching reagent are fully dissolved prior to fixing cells 9. Do not loose your pellet at the nuclei prep stage Proprietary 37

Demo success story Takeaways Date Activity March 2, 2017 Contacted Eric Nestler, MD, Ph. D Dean for Academic & Scientific Affairs, Director Friedman Brain Institute Professor | Neuroscience Professor | Pharmacological Sciences Professor | Psychiatry March 6, 2017 Contacted Philipp Mews, Ph. D Post-Doctoral Research Fellow March 15, 2017 Conference Call with Philipp Mews April 2017 Scheduled tru. Ch. IP Demo May 2017 ME 220 Sale • Identify and contact decision maker(s) • Listen and understand • Leverage Covaris Product F&B that can provide value to the end-user • Utilize Resources – Publications – Application Notes • Every opportunity is different “Our lab achieves good shearing with the Bioruptor, and we like the high-throughput setup compared to the one sample processing with the Covaris. I did use a Covaris during my graduate work with Dr. Berger at Penn, but since we got a good setup running in the Nestler lab we would only be interested in technology optimized for very low input Ch. IP. . If you think your system really does offer major advantages then I would be excited to arrange a phone call and discuss details, of course!” Philipp Mews, Ph. D Proprietary 38

Identifying new leads Pub. Med Search 1. Simple Search – Ch. IP-Seq “USA” 2. Add filters to pull papers only from research centers in the targeted region 3. Toggle Author information tab to identify investigators’ contact information 4. Begin contacting investigators via mail merger and/or automated marketing tools (see slide 20 for e-mail example) Proprietary 39

Key Search Terms – Identify Opportunities • • • Ch. IP-Seq Oncology (Breast Cancer and Ch. IP-Seq) Immunology (T-cell and Ch. IP-Seq) Primary cells and Ch. IP-Seq Mammalian cells and Ch. IP-Seq DNA methylation Methyl-Seq WGBS Ox. BS-Seq Proprietary 40

Outreach and follow-up • • E-mail campaigns Include a “call to action” in the e-mail Ask questions to determine pain points Present Covaris as a means to improve sample prep (bottleneck) workflow Proprietary 41

Identifying new leads Identify • Search Pub. Med for recent papers (focus on ones published in the last two years: >recent=better results). • A simple search term will identity hundreds of papers (e. g. , Ch. IP-Seq Japan) • Scan through the methods section. Try to focus on papers that cite using shearing with other techniques (Probe (QSONICA, Q 700), Bath (Diagenode, Bioruptor), Enzymes (Cell Signaling)) • Create a personalized message with a couple key value points (see example on slide 17) and questions • Tip: Questions help increase response rates Qualify • How many samples are you processing weekly/monthly for Ch. IP-Seq? • In the US, labs that are processing >10 samples/week are stronger leads and have funds to acquire capital equipment budgeted ~$25 -65 K USD Proprietary 42

E-mail template Hello Dr. Sun, I hope this message finds you well. My name is Sean Gerrin and I’m from the Applications Group at Covaris. Based on my literature search using Pub. Med, I understand your research involves studying transcriptional factors, histones, and other DNA-binding proteins. • Would you be interested in trying a Ch. IP sample prep method that can improve the quality and reproducibility of your NGS Library prep? • We would be happy to provide a product demonstration in your lab using your starting material as an initial evaluation. Would you be interested in a demo in November or December? We offer our tru. Ch. IP method that standardizes cell fixation, nuclei prep, and shearing for your Ch. IP-Seq protocols. To learn more from our website, please Click Here. Key Advantages • Consistent shearing bp range required for NGS Library Prep (100 -500 bp average size with the main peak between 200 -300 bp) • Lower, non-contact, and controlled energy for unbiased & random fragmentation without damage to the protein’s epitope for IP (compared to probe and bath sonicators) I look forward to receiving your feedback and seeing how we can help improve your Ch. IP sample prep workflow. Proprietary 43

Comprehensive epigenomic workflow solutions Proprietary 44