hmedip kit Hydroxymethylated DNA Immunoprecipitation USER GUIDE Cat. No. C C C FEATURES

Transcription

1 hmedip kit Hydroxymethylated DNA Immunoprecipitation Cat. No. C C C FEATURES Get your hydroxymethylation profile in 24h Includes hydroxymethylated, methylated and unmethylated DNA for controls Improved handling and reproducibility (Magnetic beads and magnetic rack) Can be used in combination with MeDIP (Dual MeDIP) Highly specific (monoclonal or polyclonal antibodies for 5-hmC) Optimized DNA isolation bufferinstruction USER GUIDE Version 3 01_2018

2 Please read this manual carefully before starting your experiment

3 PAGE 3 Contents Introduction 4 Kit Method Overview 6 Kit Materials 7 Kit Contents 7 Required Materials Not Provided 7 Kit Components 8 Protocol 11 STEP 1: Binding antibodies to hydromethylated DNA and bead washes 11 STEP 2: Magnetic immunoprecipitation and washes 13 STEP 3: DNA isolation 14 STEP 4: Quantitative PCR & Data analysis 15 Results 17 Related Products 18 Ordering Information 26

4 PAGE 4 DIAGENODE hmedip KIT MANUAL Introduction MANUAL The Diagenode hmedip kit is designed to immunoprecipitate hydroxymethylated DNA (hmethyl DNA IP). This kit is the first and only example of MeDIP kit specifically designed and fully validated for affinity-capture and detection of hydroxymethylated regions using the highly specific rat, mouse or rabbit antibodies against 5-hmC. One of the fastest growing fields in biology and cancer research is epigenetics. While the underlying genetic code defines which proteins and gene products are synthesized, it is epigenetic control that defines when and where they are expressed. Epigenetic control is generally mediated by methylation of cytosine to 5-methylC (5-mC) in CpG islands and post-translational modification of histones. Methylation of CpGs near promoters is associated with gene silencing, as is deacetylation of histones. There is substantial interest and speculation in the role of a recently discovered second type of DNA methylation, 5-hydroxymethylcytosine (5-hmC), although its precise function has not yet been elucidated. This new cytosine base modification results from the enzymatic conversion of 5-Methylcytosine into 5-Hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics: 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting hmc to C and, as such open up entirely new perspectives in epigenetic studies. Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (eg. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way and so far the only way to differentially and specifically enrich 5mC and 5hmC sequences. Recently, Diagenode launched new, highly-specific antibodies (monoclonal and polyclonal) and kits for the differential study of the functions of 5-hmC and 5-mC. In the hmedip kit, our antibody directed against 5-hydroxymethylcytosine is provided as well as hmedna, medna and undna internal IP controls. The IP has been optimized to specifically select and precipitate hydroxymethylated DNA fragments by the use of our antibodies, buffers and protocol. The IP efficiency can indeed be doublechecked with the use of our internal controls. The hmedip kits allow you to perform DNA hydroxymethylation analysis of your sample together with optimized internal IP control. Performing hydroxymethylation profiling with the hmedip kit is FAST, RELIABLE and HIGHLY SPECIFIC. In the hmedip kit, the protocol has been improved to allow researchers to work in small tubes. The kit ensures the use of low amount of reagents per reaction. The number of steps is reduced and handling is easier with our Magnetic hmethyl DNA IP procedure. The hmedip protocol is flexible, as the IP d DNA can be isolated and/or purified in different ways based on the downstream application: an extra-fast and simplified protocol is included in the hmedip kit (for qpcr analysis); but more traditional methods are also proposed (see separate module). Moreover,our Magnetic Rack together with our new hmedip kit protocol ensures the best IP conditions by working at a constantly cooler temperature. The Diagenode Magnetic Rack has been designed to be used in IP experiments, keeping samples cool longer and allowing the use of small tubes to reduce the reaction volumes and waste of reagents. The hmedip kit protocol has been validated with our Bioruptor. Nevertheless, DNA can be sheared with any in house protocol and sonication apparatus as long as efficiency is checked before use. Innovating Epigenetic Solutions

5 PAGE 5 The hmedip kit contains all reagents you need for you hmedip Assay but it can be purchased with two additional modules for: 1/ Preparation of larger quantities of genomic DNA and 2/ Traditional purification of the IP d DNA for subsequent next generation sequencing analysis. 1/ The XL GenDNA Module is optimized for the preparation of large quantities of DNA ready-to-use in Methyl DNA IP. An optimized protocol for DNA shearing is provided as well. 2/ The DNA purification includes all the reagents and buffers needed: eluting DNA from the washed beads after IP (using buffers D, E, F) and to proceed to phenol/chloroform extractions and ethanol precipitation (using DNAIP TE, DNA-IP co-precipitant and DNA-IP precipitant). Note that purification columns can also be used after elution. The hmedip kit contains all reagents you need for your hmedip Assay. Two additional products can be purchased for: 1/ Preparation of larger quantities of genomic DNA: This kit contains the GenDNA module for 6 reactions. The XL GenDNA Module (Cat. No. C ) is optimized for the preparation of larger quantities of DNA ready-to-use in Methyl DNA IP. An optimized protocol for DNA shearing is provided as well in this manual. 2/ Alternative purification method prior to next generation sequencing: The IPure kit v2 (Cat. No. C ) is the only DNA purification kit that is specifically optimized for extracting very low amounts of DNA after ChIP and MeDIP. Compatible with next generation sequencing.

6 PAGE 6 hmedip KIT MANUAL DIAGENODE MANUAL Kit Method Overview 5-hmC 5-mC 5-hmC 5-mC 5-mC Magnetic hmedip kit. 1. Cell collection and lysis 5-mC 2. DNA Extraction and Shearing using Bioruptor 5-hmC 5-mC 5-mC 3. Immunoprecipitation, washes and DNA isolation a. a. b. a. c. c. d. b. a. Add magnetic beads coated with antibody of interest b. Magnetic capture of Antibody - DNA complex c. Washes d. DNA isolation before qpcr Innovating Epigenetic Solutions b. c. d. d.

7 PAGE 7 Kit Materials Kit Contents The kit content is sufficient to perform 16 hydroxymethylated DNA Immunoprecipitations (hmedips). The kit content is described in Table 1. Upon receipt, store the components at the temperatures indicated in Table 1. Required Materials Not Provided Reagents for the IP and qpcr analysis - Gloves to wear at all steps - Autoclaved tips - RNase/DNase-free 1.5-ml (or 2-ml) tubes - PCR tubes and reagents - Ultra pure water Equipment for the IP and qpcr analysis - Diagenode Magnetic Rack (Cat. No. B ) - Centrifuges for 1.5-ml tubes (4 C) - Rotating wheel (4 C) - Vortex - Thermomixer (55 C, 95 C) - Quantitative PCR facilities Reagents and equipment for the DNA preparation and shearing - Tubes: 1.5-ml and 50-ml conical tubes - Trypsin-EDTA - Ice-cold PBS buffer - Agarose and TAE buffer - DNA molecular weight marker - Centrifuges for 1.5-ml tubes and 50-ml conical tubes (4 C) - Agarose gel apparatus - Cell counter - Bioruptor : sonication apparatus from Diagenode (Cat. No. UCD-300, website: Reagents for IP d DNA purification (option #2) - IP d DNA purification module from Diagenode (Cat. No. C ) - Phenol/chloroform/isoamyl alcohol (25:24:1) - Chloroform/isoamyl alcohol (24:1) - Ethanol 100% - Ethanol 70% - Fume hood

8 PAGE 8 DIAGENODE hmedip KIT MANUAL Kit Components MANUAL hmedip kit x16 (monoclonal rat antibody) Cat. No. C Component Comments Quantity Storage Water - 24 ml 4 C hmedip buffer H1 Ion chelator mix included (10x). 3 ml 4 C hmedna control 0,001 ng/µl 40 µl -20 C medna control 0,00025 ng/µl 40 µl -20 C undna control 0,00025 ng/µl 40 µl -20 C 5-hmC monoclonal antibody (rat) 1,6 µg/µl 32 µl (50 µg) -20 C Rat IgG 1 µg/µl 50 µl -20 C/4 C Protein G-coated magnetic beads hmedip buffer H2 The beads are supplied for 16 IPs; detergent and 0.02%, sodium azide included. BSA and Ion chelator mix included. 220 µl 4 C Do not freeze 12 ml 4 C hmedip buffer H3 Ion chelator mix included. 4 ml 4 C DNA isolation Buffer (DIB) - 5 ml 4 C Proteinase K 100 x stock solution. 40 µl - 20 C hmedna primer pairs 5 μm each (Rv & Fw). 50 µl - 20 C medna primer pairs 5 μm each (Rv & Fw). 50 µl -20 C undna primer pairs 5 μm each (Rv & Fw). 50 µl -20 C Mouse Sfi1 primer pairs 5 μm each (Rv & Fw). 50 µl -20 C PCR tube strips For 1 row of 8 samples each. 4 RT PCR tube caps For 1 row of 8 samples each. 4 RT Innovating Epigenetic Solutions

9 PAGE 9 hmedip kit x16 (monoclonal mouse antibody) Cat. No. C Component Comments Quantity Storage Water - 24 ml 4 C hmedip buffer H1 Ion chelator mix included (10x). 3 ml 4 C hmedna control 0,001 ng/µl 40 µl -20 C medna control 0,00025 ng/µl 40 µl -20 C undna control 0,00025 ng/µl 40 µl -20 C 5-hmC monoclonal antibody (mouse) 1 µg/µl 50 µl (50 µg) -20 C Mouse IgG 1 µg/µl 50 µl -20 C/4 C Anti-mouse IgG-coated magnetic beads hmedip buffer H2 The beads are supplied for 16 IPs; detergent and 0.02%, sodium azide included. BSA and Ion chelator mix included. 220 µl 4 C Do not freeze 12 ml 4 C hmedip buffer H3 Ion chelator mix included. 4 ml 4 C DNA isolation Buffer (DIB) - 5 ml 4 C Proteinase K 100 x stock solution. 40 µl - 20 C hmedna primer pairs 5 μm each (Rv & Fw). 50 µl - 20 C medna primer pairs 5 μm each (Rv & Fw). 50 µl -20 C undna primer pairs 5 μm each (Rv & Fw). 50 µl -20 C Mouse Sfi1 primer pairs 5 μm each (Rv & Fw). 50 µl -20 C PCR tube strips For 1 row of 8 samples each. 4 RT PCR tube caps For 1 row of 8 samples each. 4 RT

10 PAGE 10 DIAGENODE hmedip KIT MANUAL MANUAL hmedip kit x16 (polyclonal rabbit antibody) Cat. No. C Component Comments Quantity Storage Water - 24 ml 4 C hmedip buffer H1 Ion chelator mix included (10x). 3 ml 4 C hmedna control 0,001 ng/µl 40 µl -20 C medna control 0,00025 ng/µl 40 µl -20 C undna control 0,00025 ng/µl 40 µl -20 C 5-hmC polyclonal antibody (rabbit) crude serum 50 µl -20 C Rabbit IgG 1 µg/µl 50 µl -20 C/4 C Protein A-coated magnetic beads hmedip buffer H2 The beads are supplied for 16 IPs; detergent and 0.02%, sodium azide included. BSA and Ion chelator mix included. 220 µl 4 C Do not freeze 12 ml 4 C hmedip buffer H3 Ion chelator mix included. 4 ml 4 C DNA isolation Buffer (DIB) - 5 ml 4 C Proteinase K 100 x stock solution. 40 µl - 20 C hmedna primer pairs 5 μm each (Rv & Fw). 50 µl - 20 C medna primer pairs 5 μm each (Rv & Fw). 50 µl -20 C undna primer pairs 5 μm each (Rv & Fw). 50 µl -20 C Mouse Sfi1 primer pairs 5 μm each (Rv & Fw). 50 µl -20 C PCR tube strips For 1 row of 8 samples each. 4 RT PCR tube caps For 1 row of 8 samples each. 4 RT Innovating Epigenetic Solutions

11 PAGE 11 Remarks before starting Starting material: sheared genomic DNA Prior to hydroxymethylated DNA immunoprecipitation (hmedip), genomic DNA is first extracted from samples and then sheared. We highly recommend the use of Diagenode s GenDNA module (Cat.No. C ) for DNA extraction. It has been optimized for the preparation of genomic DNA from cultured cells. Genomic DNA must then be randomly sheared by sonication to generate fragments around 400 bp (see example below). To perform the hmedip at least 1 µg of sheared DNA is needed in a volume smaller than 100 µl. Transfer DNA sample into appropriate sonication tubes. Shear DNA by sonication using the Bioruptor. Choose the protocol and consumables which are adapted to your device. When using the Bioruptor Plus use 0.5 ml Microtubes and 0.5/0.65 ml tube holder for Bioruptor Standard & Plus & Pico and follow the protocol When using the Bioruptor Pico use 0.65 ml Microtubes (C ) Microtubes and 0.5/0.65 ml tube holder for Bioruptor Standard & Plus & Pico (B ) and follow the protocol When using 0.2 ml microtubes for Bioruptor Pico (C ) and Tube Holder 0.2 ml for Bioruptor Pico (B ) for 16 samples and follow the protocol Caution: Only use the recommended tubes! Example of shearing for hmedip using the Bioruptor Pico The genomic DNA was diluted in TE buffer to reach a concentration of 100 ng/µl and 100 µl were sheared in a 0.65 ml Bioruptor Microtube (Cat. No. C ). The following program was used: Cycles: [15 seconds "ON" & 90 seconds "OFF"] 8 cycles Agilent High Sensitivity DNA chip profile of sheared genomic DNA: smear around 400 bp

12 PAGE 12 DIAGENODE hmedip KIT MANUAL Protocol MANUAL Prior to hydroxymethylated DNA immunoprecipitation (hydroxymethyl DNA IP), DNA samples are first prepared and sheared with Diagenode XL GenDNA module (Cat. No. C ). STEP 1. Binding antibodies to hydroxymethylated DNA and bead washes 1. Prepare the IP incubation mix w/o antibodies and w/o magnetic beads for all your hmedip reactions (Table 1). Make sure when working with hmedip buffer H1, that there are no crystals left in solution. Otherwise heat up gently and mix until complete disappearence of such crystals. Table 1: IP incubation mix with no antibodies and no beads Reagent Volume per IP + INPUT Volume per additional IP Water 91.5 µl µl hmedip buffer H1 12 µl 10 µl hmedna control 1.5 µl 1.25 µl medna control 1.5 µl 1.25 µl undna control 1.5 µl 1.25 µl DNA sample (0.1 µg/µl) 12 µl 10 µl TOTAL VOLUME µl µl * If the DNA sample is at a concentration of 0.1 μg/μl, use 91.5 μl water per IP. If the concentration of the DNA sample is not at 0.1 μg/ μl, adjust the volumes of DNA and of water to add. Keep the volume of the incubation mix at 120 μl per IP+input. Caution : in any case 1 µg of DNA is needed per IP! 2. Incubate at 95ºC for 10 minutes. 3. Quickly chill sample on ice (it is best to use ice-water). 4. Perform a pulse spin to consolidate your sample. 5. First, take out 10 μl per INPUT (that is 10% input) and transfer to a new labeled tube. Keep the input samples at 4 C. The input sample is to be used as a control of starting material and it is therefore not to be used in IP. 6. Transfer from what is left: 100 μl per tube into 200-μl tubes using the provided 200-μl tube strips (or individual 200-μl tubes that can fit in our Magnetic Rack). 7. If the kit contains: a) A rat monoclonal 5-hmC antibody, add 1.6 μl of the 5-hmC antibody or 2.5 μl of Rat IgG per tube. b) A mouse monoclonal 5-hmC antibody, add 2.5 μl of the 5-hmC antibody or 2.5 μl of Mouse IgG per tube. c) A rabbit polyclonal 5-hmC antibody, add 2.5 μl of the 5-hmC antibody or 2.5 μl of Rabbit IgG per tube. The IgG is a negative control antibody. We recommend to include one IgG control for each serie of hmedip reactions. 8. Incubate on a rotating wheel (40 rpm) at 4ºC for 2 hours. During this time proceed to the beads preparation. Innovating Epigenetic Solutions

13 PAGE 13 Beads preparation 9. Wash the magnetic beads with ice-cold hmedip buffer H2 as follows: In 1.5 ml tube, add 200 μl of hmedip buffer H2 to 12 µl of beads and resuspend the beads. Incubate on a rotating wheel (40 rpm) at 4ºC for 2 hours. Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. 10 μl of beads are needed per IP. Make sure that the magnetic beads correspond to the specific 5-hmC antibody (se kit component table). 10. After washing, briefly spin the tube containing the beads to bring down liquid caught in the lid. Pellet the beads, discard the supernatant and keep the bead pellet (see below two options to pellet the beads). Options: a) Use the Diagenode Magnetic Rack (DiaMag1.5-Cat. No. B ). b) Centrifuge for 5 minutes at 1,300 rpm. 11. In a new tube, dilute 1:10 in water, the provided hmedip buffer H1 to have the diluted hmedip buffer H1: μl of hmedip buffer H1:10 are needed per IP. 12. Resuspend the beads in diluted hmedip buffer H1:10 to the volume then originally used. for 1 IP: 12 μl of hmedip buffer H1:10 STEP 2. Magnetic immunoprecipitation and washes 13. Briefly spin the 8-tube strip containing the incubation mix with antibody or isotype control (rat, mouse or rabbit IgG) (from Point 8.) and add 10 µl of washed beads per tubes (from Point 12.). 14. Incubate on a rotating wheel (40 rpm) at 4ºC for overnight. DAY Place the 8 tubes strip in the Magnetic rack (DiaMag02-Cat. No. B ), wait 1 minute and discard the buffer. 16. Wash three times using 100 μl of ice-cold hmedip buffer H1:10. Each wash is done as follows: add buffer, close the tube caps, invert the 8-tube strip to resuspend the beads, incubate for 5 minutes at 4 C on a rotating wheel (40 rpm), spin, place in the Magnetic Rack, wait 1 minute and discard the buffer. Keep the captured beads. Do not disturb the captured beads attached to the tube wall. Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning in the Diagenode Magnetic Rack. 17. Wash one time with 100 µl ice-cold hmedip buffer H3 (as described above: Point 16), spin, place in the Magnetic Rack, wait 1 minute and discard the buffer. Keep the captured beads for Point 20.

14 PAGE 14 DIAGENODE hmedip KIT MANUAL STEP 3. DNA isolation MANUAL Note: This kit includes a DNA isolation buffer for easy and very fast DNA isolation, which provides you with DNA suitable for qpcr analysis (STEP 4.) If you need DNA of higher purity for next generation sequencing or other downstream application than PCR, we suggest to use the IPure kit v2 (Cat. No. C ). Diagenode s IPure kit is the only DNA purification kit that is specifically optimized for extracting very low amounts of DNA after ChIP & MeDIP. DIB IPure v2 (Cat. No. C ) Time 30 minutes - 1h15 1h DNA concentration + ++ (possible to concentrate) DNA purity + ++ Subsequent analysis qpcr Next generation sequencing, microarray, qpcr amplification 18. Take the input samples, centrifuge briefly and from now onwards treat the input DNA samples and IP samples in parallel. 19. Prepare 100 μl complete DIB buffer per sample as follows. Add 1 μl of Proteinase K per 100 μl of DIB buffer. Scale accordingly knowing that 100 μl are needed per IP d DNA sample and 90 μl, per input DNA sample. 20. Remove the tubes from the Magnetic Rack and add 100 μl of complete DIB buffer per IP d DNA sample. Resuspend the beads and transfer the suspension into 1.5-ml tubes. 21. Add 90 μl of complete DIB buffer to 10 μl of input DNA sample. 22. Incubate at 55 C for 15 minutes both IP d DNA sample and input DNA sample. 23. Next, incubate at 100 C for 15 minutes all the samples. 24. Label new 1.5 ml tubes. 25. Centrifuge at 14,000 rpm for 5 minutes at 4 C. 26. Transfer the supernatants in new labeled tubes. That is the DNA ready for qpcr analysis. Store at -20 C. Innovating Epigenetic Solutions

15 PAGE 15 STEP 4. Quantitative PCR & Data analysis This last step consists in amplifying and analysing the IP d DNA. 27. Prepare your qpcr mix using SYBR PCR Green master mix and start out qpcr. qpcr mix (total volume of 25 μl/reaction: μl of water μl of master mix (e.g.: iq SYBR Green supermix) μl of provided primer pair (stock: 5 μm each: reverse and forward) μl of isolated DNA or INPUT PCR Amplification Temperature Time Cycle 95 C 7 minutes x1 95 C 15 seconds 60 C 60 seconds 95 C 1 minute x1 Melting Curve 65 C and increment of 0.5 C per cycle 1 minute x When the PCR is done, analyse the results. Some major advices are given below. x40 Data interpretation When the PCR is done, analyse the results. Some major advices are given below. Your own primer design - Self-complementarity and secondary structure of the primers can be tested for primer design ( wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Annealing temperature of 60oC is recommended for qpcrprimers. - Short length of amplified DNA fragment ( bp) facilitates the PCR efficiency and reduces potential problems in amplification of G/C-rich regions. - Difference in melting temperature between forward and reverse primers should not exceed 2 to 3oC. - G/C stretches at the 3 end of the primers should be avoided. Advantages of the qpcr qpcr or Real time PCR enable fast, quantitative and reliable results. info/. The Gene Quantification page describes and summarises all technical aspects involved in quantitative gene expression analysis using real-time qpcr & qrt-pcr. It presents a lot of applications, chemistries, methods, algorithms, cyclers, kits, dyes, analysis methods, meetings, workshops, and services involved.

16 PAGE 16 DIAGENODE hmedip KIT MANUAL Validation of your primers MANUAL - Test primer sets by in silico PCR: Primers should amplify unique DNA products from the genome. - Test every primer set in qpcr using 10 fold-serial dilutions of input DNA. Calculate amplification efficiency (AE) of primer set using the following by formula(1): AE= 10^(-1 / slope) - The ideal amplification factor is 2. If it is not the case the qpcr reagents from different brand or new primers should be tested. Data interpretation The efficiency of hydroxymethyl DNA immunoprecipitation of particular genomic locus can be calculated from qpcr data and reported as a percentage of starting material: % (hmedna-ip/ Total input). % (hmedna-ip/ Total input)= 2^[(Ct(10%input) ) - Ct(hmeDNA-IP)]x 100% Here 2 is the AE (amplification efficiency); Ct (hmedna-ip) and Ct (10%input) are threshold values obtained from exponential phase of qpcr for the hydroxymethyl DNA sample and input sample respectively; the compensatory factor (3.32) is used to take into account the dilution 1:10 of the input. The recovery is the % (hmedna-ip/ Total input). Background determination The final goal of IP is to calculate the enrichment in the same IP sample of: 1/ the specific DNA fragments (corresponding to the hydroxymethylated DNA) in comparison with 2/ non-methylated DNA (i.e. negative undna control). Relative occupancy can be calculated as a ratio of specific signal over background. Occupancy= % input (specific loci) / % input (background loci) Relative occupancy is then used as a measure of the hydroxymethylation of a specific locus; it provides clues about specificity of the IP. (background loci) corresponds to the signal obtained with one of the unmethylated DNA kit control. Innovating Epigenetic Solutions

17 PAGE 17 Results hmedip results obtained using the hmedip kit (mouse monoclonal antibody), including: 5-hmC mouse monoclonal antibody 5-hmC, 5-mC & cytosine DNA standard pack Mouse IgG hmedip was performed using the hmedip kit (mouse monoclonal antibody). The IgG isotype antibody from mouse was used as negative control. 1 µg of Hela cells DNA was prepared and sonicated with Bioruptor to obtain DNA fragments of bp. Unmethylated, methylated and hydroxymethylated spike-in controls have been used. Finally qpcr using specific primer pairs for the unmethylated, methylated and hydroxymethylated DNA sequences has been performed.

18 PAGE 18 DIAGENODE hmedip KIT MANUAL Related Products MANUAL BIORUPTOR SONICATION Ideal for DNA and Chromatin Shearing as well as for cell and tissue disruption. With hundreds of publications the Bioruptor has acquired an unmatched reputation in the scientific community. > Simple operating procedure > Sonication in sealed tubes > Better reproducibility of results > Validated ChIP and MeDIP protocols Note: database of shearing protocols available on our website. Diagenode s Bioruptor sonicators (Bioruptor, Bioruptor Next Gen, Bioruptor Twin, Bioruptor XL) use a unique system to uniformly process multiple samples simultaneously in sealed tubes of 0.5 ml to 50 ml capacity. It consists of a high power ultrasound generating element which is located below a water bath. The frequency of the ultrasound energy produced by the Bioruptor is 20 khz, which is similar to a probe sonicator. The Bioruptor allows you to sonicate 6 to 48 sealed tubes simultaneously without aerosol formation improving biosafety (e.g. Mycobacterium, Viruses, etc.). The continuous sample rotation guarantees equal distribution of the energy and therefore 100% reproducible results. The parameters can be efficiently controlled and allow for the automation of the sonication step in your experiments. The Bioruptor is easy to setup and uses standard disposable containers (PCR tubes, Eppendorf, 15 ml and 50 ml Falcon/Corning tubes). Validated protocols (e.g. ChIP, MeDIP etc.) can be standardized and transferred between labs. What are the effects of ultrasound on biological samples? High powered ultrasound waves can produce gaseous cavitation in liquids. Cavitation is the formation of small bubbles of dissolved gases or vapors due to the alteration of pressure in liquids. These bubbles are capable of resonance vibration and produce vigorous eddying or microstreaming. This mechanical stress has multiple effects on biological samples including; effective cell lysis, DNA and chromatin shearing as well as homogenization. When using a probe sonicator, the microstreaming phenomenon is limited to the vicinity of the probe which can generate high amounts of heat and release metal fragments. In contrast, the Bioruptor s water bath is equally exposed to ultrasound energy allowing for the dissipation of heat and providing uniform absorption of energy. Major benefits of the Bioruptor > Reproducibility > Time-saving > Automated and High Throughput > No foaming > No risk of contamination between samples > Wide range of sample size (10 µl - 20 ml) > Validated for ChIP, hmedip & libraries (next-gen sequencing) Innovating Epigenetic Solutions

19 PAGE 19 PRODUCT BIORUPTOR PLUS BIORUPTOR PICO DESCRIPTION Best suited for cell and tissue sample preparation Best suited for ChIP-seq and NGS sample preparation. KEY APPLICATIONS Chromatin shearing 200 bp - 1 kb DNA/RNA/Protein extraction Mass spectrometry Chemical applications Chromatin shearing 200bp - 1kb DNA shearing 150bp - 1kb RNA shearing 200bp - 1kb FFPE nucleic acid extraction Cell lysis and tissue disruption THROUGHPUT 12 (0.5 ml tube holder) 6 (1.5 ml tube holder) 6 (15 ml tube holder) 3 (50 ml tube holder) 12 (0.1 ml tube holder) 16 (0.2 ml tube holder) 12 (0.65 ml tube holder) 6 (1.5 ml tube holder) 6 (15 ml tube holder) RECOMMENDED VOLUMES 100 µl (0.5 ml Bioruptor tubes) µl (1.5 ml Bioruptor tubes) 300 µl - 2 ml (15 ml Bioruptor tubes) 2-20 ml (50 ml tubes) 5-50 µl (0.1 ml Bioruptor tubes) µl (0.2 ml Bioruptor tubes) 100 µl (0.65 ml Bioruptor tubes) µl (1.5 ml Bioruptor tubes) 300 µl - 2 ml (15 ml Bioruptor tubes) TEMPERATURE CONTROLLED

20 PAGE 20 DIAGENODE hmedip KIT MANUAL MANUAL Here at Diagenode we are committed to remain the best-in-class epigenetic kits supplier by meeting the epigenetic research community needs. Diagenode s kits are easy-to-use and will deliver rapid, sensitive and reproducible results. They are designed to support every step of your experiment, save you time and require minimal starting material. We have kits to perform individual steps of your experiment or cover the complete assay from start to finish. The two techniques that we are currently placing a large emphasis on are the ChIP and MeDIP assays. CHROMATIN FUNCTION Chromatin Immunoprecipitation Chromatin Immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. ChIP assay offers a huge potential to improve knowledge about the regulation of the genome expression. This technique is now used in a variety of life science disciplines and addresses several essential questions; for example cellular differentiation, tumor suppressor gene silencing as well as the effect of histone modifications on gene expression. ChIP (crosslinked) procedure Chromatin-bound proteins are formaldehyde fixed to the DNA. The chromatin is then sheared to small fragment sizes (200 bp 1 kb) and immunoprecipitated using a specific ChIP-grade antibody. Following reverse crosslinking and proteinase K treatment, the purified DNA is analyzed to identify the genomic regions that the specific protein is bound to. ChIP, ChIP-on-chip and ChIP-Seq are used to investigate interactions between proteins and DNA in vivo. It allows the identification of binding sites of DNA-binding proteins both efficiently and quantitatively. These protocols have been optimized to analyze proteins closely bound to the chromatin, including transcription factors, replicationrelated proteins, histones, histone variants and histone modifications. ChIP combined with microarray hybridization (ChIP) or sequencing (Seq) can localize protein binding sites which may help in identifying functional elements in the genome (whole-genome or specific genomic regions). All our products have been extensively validated in ChIP using various protein targets. The combination of our kits, reagents and equipment is the perfect starting point to your ChIP success. Innovating Epigenetic Solutions

21 PAGE 21 KIT ideal ChIP-qPCR ideal ChIP-seq for Histones ideal ChIP-seq for TF True MicroChIP Universal Plant ChIP-seq Starting material: cells/ip H: 100 K - 1 M TF: 4 M 100 K - 1 M 4 M 10 K K 180 K M Starting material: tissues/ip H: mg TF: 30 mg mg 30 mg g fresh weight Target: histones ü ü ü ü Target: TF ü ü ü ChIP-qPCR suitable ü ü ü ü ü FEATURES ChIP-seq suitable ü ü ü ü 1.5 hour handling time with 2 day turnaround ü ü ü ü ü Automated version available ü ü ü ü ü Control antibodies IgG and H3K4me3 IgG and CTCF IgG and H3K4me3 IgG and H3K4me3 Control PCR primers GAPDH TSS and Myoglobin exon 2 H19 and Myoglobin exon 2 GAPDH TSS and Myoglobin exon 2 Arabidopsis FLC - ATG and - Intron1 Cell lysis Cell lysis Cell lysis Cell lysis Cell lysis Included buffers and reagents Chromatin shearing IP Chromatin shearing IP Chromatin shearing IP Chromatin shearing IP Chromatin shearing IP DNA isolation DNA purification DNA purification Elution DNA purification ORDERING Manual kits C (24 rxn) Kits for IP-Star C (24 rxn) C (10 rxn) C (24 rxn) C (100 rxn) C (24 rxn) C (100 rxn) C (10 rxn) C (24 rxn) C (100 rxn) C (24 rxn) C (100 rxn) C (16 rxn) C (16 rxn) C (24 rxn) C (24 rxn) Customize your ChIP kit the way YOU want it! At Diagenode, we understand you don t always use every component from reagent kits. Now, with Diagenode s ChIP Kit Customizer, buy only the reagents that you actually need. You can still enjoy the benefits of Diagenode s carefully optimized, well-cited ChIP reagents from fixation reagents, perfected buffers and antibodies, library preparation reagents and more without purchasing a complete kit. The ChIP Kit Customizer delivers the flexibility you want with the validated and high-quality ChIP reagents you need. Log into our web site to customize your kit.

22 PAGE 22 DIAGENODE hmedip KIT MANUAL DNA METHYLATION MANUAL DNA is methylated following DNA replication and is involved in a number of biological processes including; regulation of imprinted genes, X chromosome inactivation and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions. Here at Diagenode we have developed a variety of assays and kits to facilitate the research of DNA methylation. DNA methylation and CpG islands DNA methylation is an epigenetic event that affects cell function by altering gene expression. It involves the addition of a methyl group to cytosine residues at CpG dinucleotides, a reaction that is catalyzed by DNA methyltransferase (DNMT) enzymes. The DNMT enzymes (DNMT 1, 3a or 3b) catalyses the transfer of a methyl group (CH3) from S-adenosylmethionine (SAM) to the carbon-5 position of cytosine generating 5-methyl cytosine. There are about 45,000 CpG islands in the human genome, and these are mostly located at promoters within first exons of genes. CpG islands are unmethylated in normal cells and their methylation has been associated with human disorders and epigenetic diseases. CpG islands located in the 5 region of a gene are known as molecular switches that can turn-off or turn-on the expression of the downstream gene. For the majority of genes, the CpG islands in their 5 regions are not methylated and they are ready to be expressed. In some cancers, CpG islands in the 5 regions of tumorsuppressor genes are methylated and their expressions are switched-off. For example the methylation of CpGs near tumor suppressor genes (p53 or p16) has been linked to their transcriptional silencing and can result in canerous tumors. This mechanism of gene silencing is known to be one of the major mechanisms of tumorsuppressor gene inactivation. What is the role of DNA methylation? In higher organisms from plants to humans, methylation protects DNA from endonuclease degradation and plays a critical role in regulating gene expression, making it essential for normal development and function. DNA methylation, Epigenetics and cancer The pattern of DNA methylation and histone modification(s) is critical for genome stability and controlling gene expression in the cell and plays an essential role in maintaining cellular function. The formation of cancer cells can be driven by the alteration of the DNA methylation pattern leading to the silencing of tumor suppressor genes. For example hyper-methylation at the promoter regions of BRCA1, hmlh1, p16ink4a and VHL lead to gene silencing and catastrophic consequences. On the other hand, global hypo-methylation, which can lead to chromosome instability, has also been recognized as a cause of oncogenesis. This is because it is often accompanied by region-specific hyper-methylation and therefore disrupts the balance of the cells gene expression. Preclinical and clinical studies suggest that part of the cancer-protective effects associated with several bioactive food components may relate to DNA methylation patterns (Davis and Uthus 2004). Innovating Epigenetic Solutions

23 PAGE 23 Bisulfite conversion KIT Hydroxy-/Methylated DNA Immunoprecipitation MBD based Premium Bisulfite MagMeDIP (seq) hmedip (seq) MethylCap (seq) Resolution Single nt bp bp bp FEATURES Downstream application Sanger, Pyroseq, (q)pcr NGS, qpcr NGS, qpcr NGS, qpcr Initial fragment size (bp) gdna (200) (200) (200) Input DNA (ng) > Turnaround time 1.5-2h 2-3 days 2-3 days 1-2 days BS conv. reagent ü na na na Restriction enzyme na na na na KIT CONTENT All buffers ü ü ü ü Controls ü ü All library prep reagents Indexes na na Separately in ideal Library prep C Separately in ideal Library prep C Separately in MicroPlex Library prep C , C Purification (columns/beads) ü Separately in IPure v2 C Separately in IPure v2 C Separately in IPure v2 C Beads for IP na ü ü ü Format (rxn) (Auto only) 10; Automation ü ü ü ü ORDERING Kits C C C C (mab rat) C , C (mab mouse) C , C (polyab rabbit) C , C C C

24 PAGE 24 DIAGENODE hmedip KIT MANUAL MANUAL Diagenode is recognized as the Best-in-class Epigenetic Antibody Company by EU programs, Academic labs and Biotech companies. Research continues to demonstrate the importance of histone modifications and how they can regulate gene expression. These include their interplay with histone variants, DNA methylation, transcription factors, RNA components and ATP-dependent chromatin remodeling or assembly factors. There are tremendous opportunities offered by the use of immuno-techniques like immunoprecipitation assays (especially in combination with DNA microarrays and High Throughput sequencing technologies). At Diagenode we know that a successful immunoprecipitation requires an effective, specific and high quality antibody. Therefore we focus our efforts on ensuring all of our antibodies meet our stringent quality control standards. Diagenode is involved in several European programs; often requiring us to produce high quality antibodies for the epigenetic field. This has allowed us to develop our antibody production and characterization procedures together with academic researchers. Therefore guaranteeing our antibodies meet the standards required by you; the researcher. Selection of our Antibodies The production of our antibodies is selected through a number of sources including: > Direct collaborations (including European programs) > Customer suggestions (licensing) > Research focus areas > Conferences The majority of our antibodies are developed and characterized in-house. These are complemented with high quality antibodies sourced from academic laboratories or primary manufacturers. Antibody production, characterization and quality controls at Diagenode Below is an outline of our robust antibody production procedures, which have been developed in collaboration with our academic partners. Most of our antibodies are directed against epigenetic specific targets; including histone modifications, chromatin modifying and/or interacting proteins as well as transcription factors. Diagenode s goal is to characterize every batch of antibody using established QC procedures and therefore maximizing the reproducibility of our antibody between batches. 1. Peptide design Our R&D department has developed techniques to increase the immunogenicity of our peptides to increase the titre and specificity of our antibodies. 2. Titre analysis We analyse the immune-response in the crude sera by performing pepide-elisa. 3. Affinity purification This is performed using the immunizing peptide. 4. Characterization The characterization of an antibody is the most important step of this process and we will commonly test using Peptide ELISA, Western Blot, Immunofluorescence and / or Dot Blot techniques. 5. ChIP-Grade validation Specific antibodies will be characterized using Chromatin Immunoprecipitation techniques with all our data available on our antibody datasheets. 6. Diagenode Next Generation Characterization Specific antibodies will be characterized using our inhouse Illumina Illumina Instrument (ChIP-seq) and / or through collaborative agreements using microarray approaches (ChIP-on-chip). Innovating Epigenetic Solutions

25 PAGE 25 QC flowchart (step by step) CRUDE SERA SINGLE AFFINITY PURIFIED Immune response Screening different bleeds, comparison QC purification Checking response in comparison with crude serum Fine titration Dot Blot Specificity Screening targeted cross reactions Specificity Screening targeted cross reactions Western Blot Testing global specificity Testing global specificity Immunofluorescence if applicable Immunofluorescence & ELISA inhibition if applicable Chromatin IP Screening of dilutions of different bleeds Chromatin IP Chromatin from 10,000 cell equivalents (NCCIT cells) 30 27, , , ,5 10 7,5 5 2,5 0 D0 (1:5000) D66 (1:5000) D0 (1:5000) D66 (1:5000) Chromatin IP Checking efficiency and quality of crude sera / purified Ab ChIP-chip / ChIP-seq Antibodies that have been consistent in ChIP are applied in the technique of ChIP-on-chip and ChIP-seq through collaborative aggreement.

26 PAGE 26 DIAGENODE hmedip KIT MANUAL Ordering information MANUAL Diagenode s.a. Europe, Asia & Australia LIEGE SCIENCE PARK Rue Bois Saint-Jean, Seraing - Belgium Phone: Fax: info@diagenode.com Diagenode Inc. USA 400 Morris Avenue, Suite 101 Denville, NJ Phone : Fax: info.na@diagenode.com Diagenode website: Innovating Epigenetic Solutions

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