Magna ChIP™ HT96
Contents
1. ChIPAb Phospho CREB Ser133 17 10050 ChIPAb Acetyl Histone H3 Lys4 17 675 ChIPAb Histone H3 Unmod Lys4 17 641 ChIPAb REST 17 10130 ChIPAb Trimethyl Histone H3 Lys79 17 672 ChIPAb RNA Pol II 17 600 ChIPAb CREB 17 608 ChIPAb HDAC1 17 656 ChIPAb Sox 2 clone 6F1 2 17 10032 ChIPAb Trimethyl Histone H3 Lys36 17 10034 ChIPAb EED Rabbit Poly 17 661 ChIPAb SUZ12 17 685 ChIPAb Phospho Histone H3 Ser10 17 10046 ChIPAb Histone H3 C term 17 620 ChIPAb RNA Polymerase II For a complete listing of Millipore s ChIPAb validated antibody primer sets visit www millipore com epigenetics and search ChIPAb To see all available antibodies visit www millipore com antibodies 5 Getting the Best Possible Results Using the Magna ChIP HT96 Kit The Magna ChIP HT96 protocol used in this manual is a modified version of the protocol used in our existing Magna ChIP kits This high throughput ChIP protocol was designed for efficient ChIP using lower amounts of input chromatin and antibody In addition the streamlined wash simplifies the procedure and generates more consistent ChIP results when processing a large number of samples Similar to the Magna ChIP Chromatin Immunoprecipitation kits Cat No 17 610 17 611 17 408 17 409 17 10085 and 17 10086 it is possible to perform the ChIP portion of this protocol in a single day using a shortened protocol that reduces incubation time How2. rotating platform at 50 100 rpm for two hours at 4 C During the incubation dilute the sonicated chromatin with the HT96 ChIP buffer to the cell concentration you wish to test Save 5 uL of dilute chromatin in a microcentrifuge tube and store at 20 this will be used as 5 input the f ollowing day Step 20 13 10 11 12 13 15 16 17 18 19 20 21 22 In order to get a useful standard curve during qPCR analysis the input should contain chromatin that is equivalent of 50 000 or more cells Otherwise use 5 uL of undiluted chromatin and adjust the percentage accordingly Add diluted chromatin to either a reservoir or a standard 96 well plate not provided Place chromatin in TECAN Spin down the 96 Well ChIP Plate briefly place the plate on the magnetic separator for 1 minute Remove supernatant add 100 pL dilute chromatin per well using TECAN Cover and gently shake the plate to resuspend magnetic particles and place the plate on a rotating platform at 50 100 rpm overnight at 4C Add sufficient cold HT96 ChIP buffer and HT96 Low Stringency IP Wash Buffer in separate reservoirs and place in TECAN Spin down the 96 well ChIP Plate briefly place the plate on the magnetic separator for 1 minute Remove supernatant Add 100 uL cold HT96 ChIP buffer incubate for 3 min then remove the supernatant Repeat wash with cold HT96 ChIP buffer twice as described in Step 16 Wash with HT96 Low Stringe
3. buffers supplied in the kit is sufficient to generate chromatin from up to 24 150 mm plates of cultured cells each plate providing chromatin for up to 1000 chromatin immunoprecipitations varies with cell and assay type Chromatin from other types of culture vessels can be isolated with slight modifications to the protocol Cell numbers can be scaled according to the performance of the antibody of interest to optimize highest signal to noise ratio relative to negative control mock IgG or negative location control For example Magna ChIP HT96 control antibodies can perform successful ChIP of as few as 1 X 10 HeLa cells This protocol is written for simplicity using 1 X 1 0 cells per ChIP to ensure optimal performance of the control antibodies Prepare 22 mL of 1X PBS 2 2 mL 10X PBS and 19 8 mL water for each 150 mm culture dish Store on ice This will be used for washes and needs to be ice cold Thaw the 200X Protease Inhibitor Cocktail Ill at room temperature for later use This product contains DMSO and will remain frozen below 18 4 C Add 540 uL of 3796 formaldehyde or 1100 uL of 18 5 formaldehyde directly to 20 mL of growth media to crosslink Gently swirl dish to mix Final concentration of formaldehyde is 1 Use high quality molecular biology grade formaldehyde Formaldehyde is stabilized with methanol Upon evaporation of methanol formaldehyde might form a white precipitate Do not use if white precipitate is visible in
4. kits are designed for performing 96 ChIP assays in a single experiment it is possible to conduct fewer reactions If fewer reactions are used additional 96 well plates for sample processing not provided are required The Magna ChIP HT96 kits utilize a magnetic protein A G bead blend that provides multiple advantages over single bead approaches In contrast to kits with either protein A or protein G our Magna ChIP A G beads allow use of a wider variety of antibody isotypes In addition the magnetic properties of these beads permit rapid processing of ChIP reactions with the use of a multichannel pipette and magnetic separation stand such as the Magna GriIP HT96 Rack Cat No 17 10071 or with the use of an automated liquid handler The Magna ChIP HT96 protocol has been designed and optimized for efficient immunoprecipitation and recovery of DNA Consequently in many cases a specific DNA purification product is not required for downstream qPCR This is especially true when using the Magna ChIP HT96 kit with high quality antibodies to abundant epitopes such as histone modifications and nuclear enzymes However for low abundance targets researchers may consider the use of DNA purification products such as the Agencourt amp AMPure amp XP DNA Clean beads or other small volume elution DNA purification products to concentrate the final DNA product prior to qPCR detection An optional protocol for using the Agencourt beads not supplied in combina
5. prior to sonication for analysis of unsheared DNA V For each cell concentration sonicate each tube for a fixed number of cycles allowing rests between cycles according to the instrument manufacturer s guidelines For example using a Misonix 3000 instrument and a 419 microtip probe use six 15 second pulses with 50 second intervals between pulses with power setting at 6 Keep tubes cool at all times VI Remove 1x 10 cell equivalents 20 uL 5 uL 2 uL from least to most concentrated sample of the sonicated chromatin from each condition to a fresh tube VII To all samples unsheared and sheared add ChIP Elution Buffer to a final volume of 50 uL VIII Add 1 uL Proteinase K and incubate at 62 C for 2 hour IX Load 10 pL and 20 uL on a 1 296 agarose gel with a 100 bp DNA marker Loading different amounts helps to avoid under or overloading X Observe which of the shearing conditions gives a smear of DNA in the range of 200 1000 bp See Figure 1 for an example Xl Repeat optimization of the shearing conditions if the results indicate that the resulting DNA is not in the desired size range Once optimal conditions have been determined it is advised that the user does not alter the cell concentration or volume of lysate per microcentrifuge tube for subsequent chromatin immunoprecipitation experiments 25 References Kuo MH et al Methods 1999 19 425 433 Bernstein BE et al Cell 2006 125 315 326 Cheung
6. should be stored at room temperature 18 25 C upon receipt Please see Kit Components section for specific components MAGNAO023 and MAGNAO024 Store at 20 C good for 6 months from date of receipt when reagents are stored properly ChIPAb Validated Antibodies and ChIP Qualified Antibodies For the ChIP application not all antibodies are capable of effectively precipitating chromatin Protein conformation protein interactions with other proteins or DNA and the amount of crosslinking can affect whether or not an antibody will work well in ChIP Consequently we make a distinction between what we call ChIP qualified antibodies and ChIP validated antibodies ChIP qualified or ChIP grade is a term typically used to describe any antibody previously demonstrated to work in ChIP Although not always directly tested by the supplier many consider these to be validated for ChIP For some this level of validation is sufficient However antibody performance in ChIP can vary between different lots Consequently in many cases antibodies labeled as ChIP grade fail to perform consistently from lot to lot To eliminate this concern when performing ChIP it is suggested that labs use well characterized antibodies that have been extensively evaluated for specificity proven to perform in ChIP and lot validated using ChIP An example of these types of antibodies is the ChIPAb Validated Antibody and Primer Sets ChIPAb antibodies are ri
7. the same plate if possible For positive control experiment antibody of interest is the Anti Trimethyl Histone H3 Lys4 antibody provided in the kit the locus of interest is the GAPDH promoter region primers provided and the negative control locus is the promoter region of an inactive gene not provided Negative Control Locus of Locus of Interest Locus of Interest ChIP DNA Locus Interest 1 2 Input ChIP with antibody of interest ChIP with negative control antibody IgG NIS 17 DNA Sonication ChIP DNA Should be Between 200 1000 bp in Length 500bp 100bp Figure 1 Sheared chromatin from formaldehyde crosslinked HeLa cells was prepared by following Section A I all Steps Section B Steps 1 5 and Appendix A of the EZ Magna ChIP A G Chromatin Immunoprecipitation Kit protocol cat 17 10086 20 uL sheared chromatin lane 2 was then electrophoresed through a 296 agarose gel and stained with ethidium bromide Lane 2 shows that majority of the DNA has been sheared to a length between 200 bp and 1000 bp Performance of the Magna ChIP HT96 Protocol Using Various Antibodies and Quantities of Chromatin in Automated ChIP 96HT ChIP Using 100 000 HeLa Cell Equivalents 2 15 c 1 2 0 9 2 o 0 6 0 3 0 0 a Antibody 96HT ChIP Using 10 000 HeLa Cell Equivalents 5 2 1 2 0 9 O 0 6 oO 0 3 c 0 0 RNAP2 pCREB IgG CTD S133 Control Antibod
8. type cell concentration and the specific sonicator equipment including the power settings and duration and number of pulses Approaches for optimizing sonication may include the following A Varying the concentration of cell equivalents per mL of initial HT96 Nuclei Isolation Buffer with constant sonication parameters B Choosing a fixed concentration of cell equivalents per mL of HT96 Nuclei Isolation Buffer and varying cycles and or power settings of sonication C A combination of both approaches The protocol presented below describes optimization following option A and is provided as an example only l Generate a cell lysate by following Section B but vary the HT96 Nuclei Isolation Buffer volume per cell amount in Step 3 to generate 3 different microcentrifuge tubes containing several cell equivalent concentrations in the range of 5 x 10 per mL to 5 x 10 per mL For HeLa cells this requires approximately 4 x 10 cell equivalents or approximately four 15 cm plates I Continue following the Cell Lysis procedure through Step 8 Each microcentrifuge tube should contain approximately 500 uL of cell lysate Volume of Cell Lysis Buffer Cell Density Cells required 500 uL 5 x 108 mL 2 5 x 10 500 uL 2 x 10 mL 1x 107 500 uL 5 x 10 mL 2 5 x 107 Il Be sure to keep the samples on wet ice at all times Sonication generates heat which will denature the chromatin IV Remove 1 x 10 cell equivalents from each condition
9. 1 min 15 G Data Analysis There are many algorithms to analyze ChIP result the two most common methods are the relative standard curve method and the AACt method l Normalize DNA concentration to percent of input using relative standard curve 1 Perform a serial dilution with the 5 input sample use more input if input concentration is low perform quantitative real time PCR with these input samples ChIP DNA samples and control samples IgG non immunized serum or no antibody control 2 Calculate the threshold cycle Ct values using real time detection system software from qPCR equipment manufactory Use the threshold cycle Ct values of these input samples to build a standard curve Determine the concentration C of the ChIP DNA as percent of input using the standard curve Determine the fold enrichment by calculating the ratio of Cap of interest ANd Ciga HE UE For each independent experiment we suggest that you perform the following ChIP qPCR assays in triplicates in the same plate if possible For positive control experiment antibody of interest is the Anti Trimethyl Histone H3 Lys4 antibody provided in the kit the locus of interest is the Gapdh promoter region primers provided and the negative control locus is the promoter region of an inactive gene not provided Negative Control Locus of Locus of Interest Locus of Interest ChIP DNA Locus Interest 1 2 Input dilution series 1 X X X Input dilut
10. Carefully remove supernatant and make sure there are no Wasnmmng Aspiration of the beads in the supernatant prior to removing it beads during buffer f j removal Use rack with magnets capable of firmly holding beads in place e g Magna GrIP Rack Cat No 20 400 When performing elution make sure that the temperature is Incomplete elution near 60 C Proteinase K will be inactivated by prolonged Elution and incubation at temperatures above 65 C Reversal of Excessive cross linking may not be reversible Conduct a crosslinking Excessive time course at a fixed formaldehyde concentration and or Crosslinking investigate a range of formaldehyde concentrations for a fixed time Ensure amplification reaction program is correctly set on Incorrect Annealing Lie eee Temperature or Re examine primers for correct Tm PUE Perform PCR on genomic DNA to confirm amplification conditions and ability of primers to generate a single DNA product of the expected size PCR Bad primers Redesign primer Increase amount of DNA added to the PCR reaction No PCR product et Increase the number of cycles for the amplification reaction l Decrease amount of DNA added to the PCR reaction PCR product is a l smear Use HotStart V Taq polymerase to avoid non specific annealing of primers 24 Appendix A Optimization of DNA Sonication Optimal conditions for shearing cross linked DNA to 200 1000 base pairs in length depend on the cell
11. Cat No 71091 e Vortex mixer e dNTPs 2 5 mM each e g Novagen 10 e Rotating end over end wheel platform e g Labnet mM dNTP Mix Cat No 71004 Mini LabRoller rotator e SYBR Green Master Mix for qPCR or e Microcentrifuge stock of SYBR Green for blending into e Sonicator qPCR reaction e Thermomixer and adapter for 96 well plate DNase and RNase free sterile H2O e Variable temperature water bath or incubator e 2 Agarose gel e Timer e 50 bp DNA Ladder e g NEB Cat No e Variable volume 5 1000 uL pipettors tips N3236S e Cell scraper e 6X gel Loading buffer e Microcentrifuge tubes 1 5 mL e DNase and RNase free sterile H2O e g e Thermal cycler Millipore Nuclease free water Cat No e Real time PCR Instrument 3098 e Filter tip pipette tips Hazards o Wear gloves when using this product Avoid skin contact or ingestion of all reagents and chemicals used in this protocol o Protease Inhibitor Cocktail III contains DMSO avoid contact with skin o Chromatin preparation may require use of liquid nitrogen Use personal protective equipment PPE when handling liquid N2 to avoid burns o Use PPE fume hoods and venting when working with concentrated formaldehyde solutions Formaldehyde is toxic by inhalation skin contact and ingestion Storage and Stability MAGNAO022 Store at 2C 8 C good for 6 months from date of receipt when reagents are stored properly Please note Some components provided in this kit
12. Chromatin i inati Sharin Denaturalletef Keep the sample on ice during sonication Shorten the time 9 proteins from of each sonication and increase the number of times the overheating sample sample is sonicated Allow sufficient time for sample to cool between pulses Choose an antibody directed to a different epitope of the recognize protein in TOR i fixed chromatin Decrease the amount of formaldehyde or fixation time in formaldehyde Not enough or too Perform IP from a dilution series of antibody with a fixed much chromatin amount of chromatin or vice versa T Incubate the antibody of interest with the chromatin at 4C Primary SEE i Vy T Antibody Insufficient incubation Select a different antibody with higher affinity time Perform a Western blot of the immunoprecipitated protein to verify that the antibody can precipitate the antigen of interest Primary antibody is Use a bridge antibody or secondary antibody compatible not compatible with wih AC beads A G beads Plate Leaks Ensure that the top of the plate is dry before applying seals Aus The magnetic beads settle to the bottom of the tube over Rea nig Not enough beads time Be sure the Protein A G magnetic beads are well Pn A mixed prior to removing the appropriate volume for IP 23 ChIP Optimization and Troubleshooting Continued Potential Step Problems Experimental Suggestions RUE WERE Increase number of washes for each wash buffer TR
13. I et al Proc Natl Acad Sci US A 2010 107 8824 8829 Soloman My et al Cell 1988 53 937 947 Oia TS Das PM et al Biotechniques 2004 37 961 969 Warranty EMD Millipore Corporation EMD Millipore warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products EMD MILLIPORE MAKES NO OTHER WARRANTY EXPRESSED OR IMPLIED THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE The warranty provided herein and the data specifications and descriptions of EMD Millipore products appearing in EMD Millipore s published catalogues and product literature may not be altered except by express written agreement signed by an officer of EMD Millipore Representations oral or written which are inconsistent with this warranty or such publications are not authorized and if given should not be relied upon In the event of a breach of the foregoing warranty EMD Millipore Corporation s sole obligation shall be to repair or replace at its option the applicable product or part thereof provided the customer notifies EMD Millipore Corporation promptly of any such breach If after exercising reasonable efforts EMD Millipore Corporation is unable to repair or replace the product or part then EDM Millipore shall refund to the Company all monies paid for such applicable Product EMD MILLIPORE CORPORATIO
14. Magna ChIP HT96 Cat No 17 10077 EZ Magna ChIP HT96 Cat No 17 10078 High Throughput Chromatin Immunoprecipitation Kit FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA amp Canada Phone 1 800 645 5476 In Europe please contact Customer Service France 0825 045 645 Spain 901 516 645 Option 1 Germany 01805 045 645 Italy 848 845 645 United Kingdom 0870 900 46 45 For other locations across the world please visit www millipore com offices Introduction Chromatin in eukaryotic cells is associated with a plethora of structural enzymatic and regulatory proteins which may interact with regional DNA sequences and affect genomic functions This dynamic and coordinated interaction may influence the expression and cellular utilization of each gene locus whether coding or non coding Thus it is crucial to elucidate DNA protein interactions in order to decipher the nuclear mechanisms underlying a wide variety of biological processes and disease states ChIP chromatin immunoprecipitation is a powerful technique classically used for mapping the in vivo distribution of proteins associated with chromosomal DNA These proteins can be histone subunits transcription factors or other regulatory or structural proteins bound either directly or indirectly to DNA Successful ChIP requires high quality ChIP validated antibodies that can specifically detect proteins associated with regions of chromosomal DNA Tradition
15. N SHALL NOT BE LIABLE FOR CONSEQUENTIAL INCIDENTAL SPECIAL OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS SYBR is a trademark of Invitrogen Corporation Agencourt amp AMPure are registered trademarks of Agencourt Biosciences Thermomixer is a registered trademark of Eppendorf AG Unless otherwise stated in our catalog or other company documentation accompanying the product s our products are intended for research use only and are not to be used for any other purpose which includes but is not limited to unauthorized commercial uses in vitro diagnostic uses ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals c 2009 2012 Merck KGaA Darmstadt All rights reserved No part of these works may be reproduced in any form without permission in writing 26 MAGNACHIPHT96MAN July 2012 Revision D
16. Using Magna ChIP HT96 Panel Il 2500 t 1935 2000 E c 1500 1219 uj 1000 U 627 O 500 0 m mH T T o Hd 3 4 33 0 O0 O OQ pzzrzzzr o o o o a 59202975 254295p SES 8 2285 3 NAY EnD x gt EI EQ P g fi N to o T R Figure 5 Chromatin was derived from sources as indicated and Mock vs Specific antibody immunoprecipitations were performed using the multichannel protocol of Magna ChlP HT96 on various loci as indicated on ChlPab product data sheets See www millipore com for details of ChiPab assays 22 ChIP Optimization and Troubleshooting Potential Step Problems Experimental Suggestions The amount of formaldehyde and time of crosslinking must be determined empirically Conduct a time course at a fixed E Not enough or too formaldehyde concentration and or investigate a range of 9 much crosslinking formaldehyde concentrations for a fixed time HINT Histones may not need to be cross linked since they are tightly associated with DNA It is important to have sufficient HT96 Nuclei Isolation Buffer T for the number of cells processed Follow the guidelines in Cell Lysis WENT CIE Feuele this protocol Also verify cell lysis by viewing a 10 uL of cells portion of the cell lysate under the microscope to confirm lack of intact cells Not enough too much Try to optimize the sonication condition to obtain the sonication appropriate DNA size
17. ally endpoint and quantitative PCR qPCR is performed after ChIP to verify whether a particular DNA sequence the gene or region of the genome is associated with the protein of interest Using this classical approach researchers can evaluate the interactions of the proteins of interest with a limited number of known target genes Recently the need has grown to map characterize and understand protein DNA interactions across the epigenome This can be done for multiple marks at a given locus as well as across the genome The need to profile these interactions across the genome has led to the development of genome wide ChIP analyses with either microarrays ChIP chip or next generation sequencing ChIP seq T Genome wide mapping of protein DNA interactions and epigenetic marks helps to elucidate mechanisms of transcriptional control and maintenance of chromatic and genomic DNA integrity within a cell In certain cases the profile of this epigenome can be used to assess progression through differentiation distinguish between normal and disease states or predict responses to chemoprevention For many laboratories ChIP can be technically demanding as it requires robust antibodies high quality reagents and careful planning and handling The Magna ChIP HT96 kit was designed to address the technical challenges when processing a larger number of samples The protocol and reagents provided simplify the standard ChIP procedure improve the reproduc
18. d a negative control location region Input DNA is required whether using relative standard curve method or the comparative Ct AACt method An example of good fold enrichment is shown in Figure 2 1 Add 2 uL of the sample to a PCR plate suitable for your real time instrument of choice not supplied 2 uL or less ChIP DNA is recommended for a 20 uL PCR reaction Performing triplicate of qPCR reactions per ChIP sample is also recommended If using the relative standard curve method perform 3 4 5 or 10 fold serial dilutions using the reverse crosslinked DNA from the 5 input sample and use these samples to build a standard curve Concentration of the ChIP samples can be calculated as percent of input using the standard curve Alternatively data can be calculated in relation to cell equivalents of chromatin or mass of purified DNA if desired Prepare a qPCR mastermix as shown in Table Prepare a sufficient volume of mastermix for one extra tube to account for pipette carryover Add 23 uL of qPCR mastermix to the 2 uL of the sample Use caps or an optical tape to seal the plate and start the qPCR reactions Please refer to Figure 2 for real time PCR result Table I qPCR reagent setup and running parameters qPCR reagent assembly for 1 reaction qPCR parameters ddH O 9 5 wl Initial Denature 94 C 10 min SYBR Green Master Mix 12 5 ul Primer mix 1 ul Denature 94 C 20 sec l Total 23 yl l Ee times Anneal and Extension 60 C
19. eads CS207285 1 1 mL HT96 Low Stringency IP Wash Buffer CS207284 30 mL HT96 ChIP Elution Buffer CS207288 20 mL Store the Following at Room Temperature Upon Receipt 96 well ChIP Plate CS209289 2 96 well Thermal Plate CS207290 1 Plate seal B 3 Strip caps 12 MAGNAO023 20 C Store at 20 C Upon Receipt Component Catalog Quantity Protease Inhibitor Cocktail Ill Animal Free 535140 1ML 1 0 mL Contains DMSO Proteinase K Solution 600mAU mL CS207286 0 2 mL MAGNAO024 20 C Store at 20 C Upon Receipt Component Catalog Quantity Protease Inhibitor Cocktail Ill Animal Free 535140 1ML 1 0 mL Contains DMSO Proteinase K Solution 600 mAU mL CS207286 0 2 mL Anti Trimethyl Histone H3 Lys4 CS200580 75 uL Normal Rabbit IgG CS200581 75 uL GAPDH primers 22 004 75 uL Functional Validation Magna ChlP HT96 modules are functionally tested in quantitative ChIP reactions to ensure quality control of the supplied components 3 Materials Required But Not Supplied Reagents Equipment e Cells stimulated or treated as desired e Microscope and cell counter e Antibody of interest for chromatin e Magnetic Separator for microcentrifuge tubes immunoprecipitation see page 6 Magna GrIP Rack 8 Well Cat No 20 400 e 37 Formaldehyde e Magnetic Separator for 96 well plate e Tag DNA polymerase e g NovaTaq Hot Magna GrIP M HT96 Rack Cat No 17 10071 Start DNA Polymerase
20. ever for some antibodies this shorter protocol can result in slightly reduced ChIP efficiency Regardless of your approach to ensure the best possible results advance planning is advised It is strongly recommended that you read the entire protocol before performing this procedure especially if you are using this kit for the first time A general overview of the major steps of a typical Magna ChIP HT96 workflow is provided on page 9 The detailed protocols and guidelines presented here will help you to avoid common pitfalls It is critical to review and follow the suggestions in the Experimental Considerations section It is also important to take the time to evaluate the samples being prepared after key steps in the protocol This will save time and materials and minimize the potential for sub optimal results Important Information for Processing Partial Plates The Magna ChIP HT96 kit provides sufficient reagents to run fewer than 96 reactions in three separate experiments To run these partial plates it is strongly recommended that new 96 well ChIP and 96 well Thermal plates with new plate seals or caps are used see below for ordering information New plates minimize the risk of cross contamination of samples and confounding data in sensitive endpoint analyses such as qPCR Ordering Information Additional 96 well plates for running partial plates Description Catalog Components 96 well ChIP plates 2 96 well Ther
21. gnetic particles that may have settled Add DNA purification beads according to the solution volume chart below DNA purification beads Volume uL 8 F Mix reagent and ChIP DNA thoroughly by pipette mixing 10 times in a standard 96 well plate not supplied Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery Place the reaction plate onto a Magna GriIP Rack 96 well for 2 minutes to separate beads from the solution Aspirate the cleared solution from the reaction plate and discard Keep the plate on the magnetic stand dispense 200 uL of 70 ethanol to each well of the reaction plate and incubate for 30 seconds at room temperature Aspirate the ethanol and discard Repeat for a total of two washes Off the magnet plate add 40 uL of water to each well of the reaction plate and pipette mix 10 times Place the reaction plate onto a Magna GrIP Rack 96 well for 1 minute to separate beads from the solution Transfer the eluant to a new plate not supplied Real Time Quantitative PCR to Verify Chip DNA Enrichment The success of ChIP can be evaluated through qPCR Verification of ChIP enrichment can be performed using the relative standard curve method of qPCR analysis to compare DNA from a mock IP vs DNA immunoprecipitated using your ChIP antibody or can alternatively be compared using relative standard curve with two PCR amplicons a positive control binding region an
22. gorously validated to ensure specificity and their ability to immunoprecipitate chromatin In addition each and every lot of a ChIPAb antibody is subject to extensive quality control testing including testing in the ChIP application ChIPAb antibodies are more than just a highly validated antibody To allow independent verification of performance or for use as a positive control all ChIPAb antibodies include a negative control IgG plus PCR primers directed against a known positive locus A partial list of ChIPAb antibodies is given in the table below Catalog Catalog Number Description Number Description 17 622 ChIPAb Trimethyl Histone H3 Lys27 17 10044 ChIPAb CTCF 17 614 ChIPAb Trimethyl Histone H3 Lys4 17 681 ChIPAb Dimethyl Histone H3 Lys9 17 658 ChIPAb Acetyl Histone H3 Lys9 17 630 ChIPAb Acetyl Histone H4 17 625 ChIPAb Trimethyl Histone H3 Lys9 17 613 ChIPAb p53 17 648 ChIPAb Dimethyl Histone H3 Lys9 17 603 ChIPAb ER 17 601 ChIPAb Sp1 17 643 ChIPAb Monomethyl Histone H3 Lys27 17 662 ChIPAb EZH2 clone AC22 17 10048 ChIPAb Histone H2A Z 17 615 ChIPAb Acetyl Histone H3 17 10054 ChIPAb Histone H2B 17 663 ChIPAb EED 17 10057 ChIPAb SMRT 17 678 ChIPAb Trimethyl Histone H3 Lys4 17 10045 ChIPAb Acetyl Histone H4 Lys5 17 677 ChIPAb Dimethyl Histone H3 Lys4 17 10098 ChIPAb TATA Binding Protein TBP 17 10051 ChIPAb Acetyl Histone H3 Lys14 17 10131
23. ibility and robustness of the ChIP process and minimize the hands on steps in ChIP This kit allows ChIP to be done in a 96 well plate format Researchers using this approach can perform up to 96 ChIP assays at once Processing of samples can be performed using a multichannel pipette or automated at multiple steps using standard liquid handlers T For details on genome wide analysis using microarrays please refer to the Magna ChIP Microarray kit user manual Cat No 17 1000 17 1001 or 17 1002 tt For details on genome wide analysis using ChIP seq please refer to the Magna ChIP Seq Chromatin Immunoprecipitation and Next Generation Sequencing Library Preparation Kit user manual Cat No 17 1010 Kit Overview The Magna ChIP HT96 kit provides a complete set of validated quality controlled reagents and a detailed protocol with in process quality control guidelines Similar to the Magna ChIP HT96 kit the EZ Magna ChIP HT96 kit contains all of the materials required for high throughput ChIP plus positive and negative control antibodies and PCR primers that can be used as in process controls or for verification of technique The materials provided in either kit allow the performance of up to 96 ChIP assays with as little as 10 000 cell equivalents of chromatin when exploring abundant epitopes and as little as 100 000 cell equivalents when measuring less abundant proteins such as sequence specific transcription factors Although these
24. ion series 2 X X X Input dilution series 3 Input dilution series 4 ChIP with antibody of interest ChIP with negative control antibody IgG NIS Il AACt method 1 Perform quantitative real time PCR with 2uL of ChIP DNA and input DNA in triplicates 2 Perform quantitative real time PCR with primer set targeting a positive locus and primer set targeting a negative locus separately 3 Calculate the threshold cycle Ct values using real time detection system software from qPCR equipment manufactory 4 Normalize ChIP DNA Ct values to input ACt for each primer set by subtracting the Ct value obtained for the input DNA from the Ct value for ChIP DNA ACt Ctcup Ctinpur Log2 Input Dilution Factor 16 Calculate the percent of input for each ChIP 9eInput 2 C normalized ChIP Normalize positive locus ACt values to negative locus AACt by subtracting the ACt value obtained for the positive locus from the ACt value for negative locus AACt ACtposiive ACthegative 7 Estimate the fold enrichment of the positive locus sequence in ChIP DNA over the negative locus Fold enrichment 244 This estimate is accurate only if the primer efficiency of both primer sets is identical and preferably 100 efficient so careful design and validation of the primer sets are essential For each independent experiment we suggest that you perform the following ChIP qPCR assays in triplicates in
25. mal plate 1 Plate seals 3 Strip caps 12 96 well Thermal Plate 1 Strip caps 12 96 well ChIP plates 2 Plate seals 3 Magna ChIP HT96 ChIP and Thermal Plate Set 17 10457 Magna ChIP HT96 Thermal Plate Set 17 10458 Magna ChIP HT96 ChIP Plate Set 17 10459 Chromatin Immunoprecipitation Experimental Considerations ChIP validated antibodies are perhaps the most important component of a ChIP experiment Ensure that the antibody being used has been validated in ChIP using genomic locations of both predicted high and low occupancy Secondly ensure that the quality of the chromatin being prepared is suitable in functional ChIP assays using control antibodies where possible and by testing a range of fragmentation by agarose gel electrophoresis If you wish to check your technique or have a source of controls you can utilize the validated positive and negative control antibodies and qPCR assay provided in the EZ ChIP kit or consider using a ChIPAb validated antibody see page 6 and qPCR primer set Chromatin size is critical to the success of ChIP This protocol works best when the chromatin size is between 200 1000 bp Shearing of chromatin varies greatly depending on cell type growth conditions quantity volume crosslinking and equipment It may be necessary to optimize sonication conditions by changing the power settings cycle number and ratios of time ON and time OFF The quality of the chro
26. matin should be evaluated by agarose gel electrophoresis of Proteinase K digested crosslink reversed purified DNA fragments Cell number equivalents of chromatin required per ChIP reaction is dependent on the quantity of available epitopes in the cell of interest as well as the quality of antibody used Successful enrichment can be performed with as low as 1X10 cells per ChIP when utilizing high quality antibodies directed towards abundant epitopes such as several histone modifications and RNA Polymerase ll It is recommended to increase the amount of cells when the antibody is less optimal or the number of epitopes per cell is lower The Magna ChIP HT96 approach used in this manual recommends preparing chromatin in batch format for approximately 10 ChIP samples per preparation with 1 000 000 cultured cells or for approximately 10 ChIP samples per preparation with 50 mg of tissue samples Sufficient buffers for chromatin preparation are provided to enable chromatin preparation for up to 24 samples Thus 240 reactions can be generated depending upon cell number e g 100 000 cells ChIP but 96 independent chromatin preps are not possible with the reagents as provided qPCR is typically used to evaluate the success of ChIP A mock IgG or negative antibody control ChIP reaction may be performed to determine fold enrichment relative to a specific ChIP antibody Alternatively a negative locus control may also be used for normalization to minimize chroma
27. ments was verified by qPCR using control primers flanking the human GAPDH promoter region 20 Well to well Carryover Contamination Using Automated Protocol 1 0 0 8 0 6 0 4 0 2 0 0 Percent of Input Well to Well Carryover Contamination Including Antibody Plate Column 1 No Antibody Plate Column 2 No Antibody Plate Column 3 B H3K4Me3 El Control IgG R o pCREB S133 o Control IgG M Figure 4 Sonicated chromatin prepared from 100 000 untreated HeLa cells was subjected to chromatin immunoprecipitation using 1 ug of purified IgG mouse IgG 12 371B Rabbit IgG 12 370 or specific antibodies H3K4Me3 17 614 Phospho CREB 17 10131 and the Magna ChIP HT96 Kit using a Freedom EVO robotic workstation Immunoprecipitation of antibody associated DNA fragments was verified by qPCR using control primers flanking the human GAPDH promoter region Standard ChIP was performed in the first column of a 96 well plate Mock IP without antibody was performed in the second and third column 21 Performance of Various ChlPab Antibodies Using Magna HT96 Antibody Performance Using Magna ChIP HT96 Panel I 47 27 28 29 31 18 12 Fold Enrichment e m M 888 DE aX _ M 2S S S 8 8 o o gt O0 0 o oc zr mncrlirscr E om 9 9 9 9 o ok y 5 X 3 42 ON mcm o 9 9S a ou Fae 5 o qt a d AN cm IL e eo r Antibody Performance
28. ncy IP Wash Buffer twice as before Step 16 during the second wash transfer the beads mix to the 96 well Thermal Plate Place the 96 well Thermal Plate on the magnetic separator for 1 minute and remove the supernatant Resuspend beads in 50 uL HT96 ChIP Elution Buffer and add 1 uL Proteinase K Itis useful to set up a mastermix so that Proteinase K is equal in each sample Add 45 uL HT96 ChIP Elution Buffer and 1 uL Proteinase K to input see Step 9 Cover the 96 well Thermal Plate with strip caps incubate the plate and input in a Thermomixer at 65 for 2 hours then at 95 for 15 minutes Al low the plate to cool down to room temperature Please use the supplied strip caps in the step The plastic plate seal does not seal properly during long periods of heating Place the 96 well Thermal Plate on the magnetic separator for 1 minute and add 45 uL of the supernatant to a new 96 well ChIP Plate be careful not to transfer the beads E DNA Purification Optional DNA purification is not required for qPCR analysis but is required for some downstream applications such as ChIP chip and ChIlP seq DNA purification allows detection of lower concentration immunoprecipitates associated with some DNA binding proteins We recommend Beckman Agencourt AMPure XP DNA purification system Cat No A63880 if DNA purification is required for your analysis 1 Gently shake the Agencourt AMPure XP DNA purification beads bottle to resuspend any ma
29. ommended that new plates be used to minimize potential cross contamination of samples Additional 96 well ChIP plates 96 well Thermal plates plate seals and strip caps are available please see page 6 for further details and ordering information l Chromatin immunoprecipitation using a multichannel pipettor 1 Gently shake the Magna ChIP A G Magnetic Beads tube to resuspend any magnetic particles that may have settled Dispense the appropriate volume of Magna ChIP A G Magnetic Beads 10 uL per ChIP reaction to a microcentrifuge tube then place the tube on a magnetic separator for 1 minute Ifa microcentrifuge magnetic rack is not available a single pin of the Magna GrIP rack 96 well can be used for this purpose 11 11 12 13 14 15 Remove supernatant Add 5 beads volume of HT96 ChIP buffer 5 times the volume of bead used then remove tube from the magnetic separator and mix the beads by gently pipette mixing several times to completely resuspend beads Place the tube on the magnetic separator for 1 minute Repeat Step 2 and remove supernatant Resuspend beads in one bead volume of HT96 ChIP buffer For multipipette application a trough may be used for distribution of 10 uL bead volumes Include sufficient overage to enable multichannel pipetting In the 96 well ChIP Plate add 90 uL HT96 ChIP buffer see note below 10 uL beads and the appropriate quantity of antibody per well Final volume should be 100 uL F
30. or 96 reactions this would be 960 uL The amount of antibody used per ChIP must be empirically determined In general 1 10 ug of purified antibodies is generally sufficient for standard immunoprecipitations For Magna ChIP HT96 3 uL of the Anti Trimethyl Histone H3 Lys4 control antibody CS207358 is sufficient for 100 000 cells and as little as 1 uL can be used for fewer cells The amount of antibody used per reaction may require optimization and in general lower amounts of antibody are appropriate for Magna ChIP HT96 reactions Itis recommended to perform a negative control ChIP using normal IgG or no antibody tis also a good idea to include technical replicates in your experiment Cover and gently shake the plate to resuspend magnetic particles then place the plate on an end over end rotator at 50 100 rpm for two hours at 4C to keep beads from settling out of solution Ensure that the top of the plate is dry before applying the plastic seal seal tightly Ensure end over end rotation in mixing During the incubation dilute the sonicated chromatin with the HT96 ChIP buffer to the cell concentration you wish to test For example if you are performing 10 ChIP reactions each with 100 000 cells then add 1 000 000 cell equivalent of chromatin to 1 mL of ChIP buffer then add 100 uL of diluted chromatin to each well You may need to prepare extra if you are using a multichannel pipette and reservoir Save 5 uL of dil
31. ove the supernatant Resuspend beads in 50 uL HT96 ChIP Elution Buffer without protein inhibitor cocktail and add 1 uL Proteinase K Add proteinase K to elution buffer to make a stock solution and use a multichannel pipette to dispense the stock solution Add 45 uL HT96 ChIP Elution Buffer and 1 uL Proteinase K to input see Step 7 Cover the 96 well Thermal Plate with strip caps incubate the plate and input in a Thermomixer at 65 C for 2 hours and then at 95 C for 15 minutes Let the plate cool to room temperature Please use the supplied strip caps in this step The plastic plate seal does not seal properly during long periods of heating Place the 96 well Thermal Plate on the magnetic separator for 1 minute then transfer 45 uL of the supernatant to a new 96 well ChIP Plate be careful not to transfer the beads ll Automated Chromatin Immunoprecipitation The protocol below was done using the TECAN Freedom EVO robotic workstation This approach can be used as a guide to using other automated liquid handing systems for appropriate steps in chromatin immunoprecipitation 1 Gently shake the Magna ChIP A G Magnetic Beads tube to resuspend any magnetic particles that may have settled Add the appropriate amount of Magna ChIP A G Magnetic Beads 10 uL ChIP to a microcentrifuge tube then place the tube on a magnetic separator for 1 minute Remove supernatant Add one bead volume of HT96 ChIP buffer remove tube from the magnetic
32. rmalin fixed paraffin embedded FFPE material can be used to obtain a small sample of interest please see the Magna ChIP G Tissue Kit manual Cat No 17 20000 A pea size mass of tissue contains around 10 cells and should be sufficient for 100 ChIP samples Specimens should be handled carefully and promptly to preserve specimen integrity Weigh the tissue and then transfer into a 50 mL tube and wash twice with ice cold 1X PBS Resuspend tissue in 20 mL ice cold PBS and add 540 uL of 37 formaldehyde or 1100 uL of 18 596 formaldehyde to crosslink Gently swirl dish to mix Incubate at room temperature for 10 minutes In the interim prepare 1X protease inhibitor in PBS Add 2 mL of ice cold 1X PBS to a separate tube for every sample and add 10 uL of Protease Inhibitor Cocktail Ill Store on ice Add 2 mL of 10X glycine to quench excess formaldehyde Homogenize the tissues several times using a Dounce homogenizer loose pestle Spin at 800 x g at 4 C for 5 minutes to pellet cells B Cell Lysis to Release Cross Linked Proteins DNA 1 OT ape ac m In the interim prepare 0 5 mL of HT96 Nuclei Isolation Buffer containing 2 5 uL of 200X Protease Inhibitor Cocktail IIl for each microcentrifuge tube Remove supernatant Cell pellet can be frozen at 80 C at this step Resuspend cell pellet in HT96 Nuclei Isolation Buffer prepared in Step 1 Incubate on ice for 15 minutes vortex the cell suspension briefly every 5 minu
33. rument operation An example of sonicated HeLa cell chromatin fractionated suitably for use with Magna ChIP HT96 is shown in Figure 1 Keep cell lysate ice cold Sonication produces heat which can denature the chromatin Allow at least 30 seconds between cycles of sonication to prevent sample overheating Spin at a minimum of 10 000 x g but not exceeding 15 000 x g at 4 C for 10 minutes to remove insoluble material Prepare a 5 uL aliquot for agarose gel analysis of the sheared DNA according to the protocol in Appendix A Steps VII to X Store on ice if gel analysis will be done in the same day otherwise store aliquot at 20 C It is important to do this step to ensure chromatin is sheared to appropriate size Remove supernatant and place 50 ul aliquots into new microcentrifuge tubes Each 50 uL aliquot contains 1 x 10 cell equivalents of lysate which is enough for up to 10 immunoprecipitations Sheared crosslinked chromatin can be stored at 80 C for up to 3 months Immunoprecipitation IP of Crosslinked Protein DNA Prior to starting this section Remove 200X Protease Inhibitor Cocktail Ill and thaw at room temperature This product contains DMSO and will remain frozen below 18 4 C Always add protease inhibitors to all buffers before use unless directed otherwise All buffers should be chilled on ice before use If using an automated workstation see Section D II To run partial plates it is strongly rec
34. separator and mix the beads by gently pipetting several times to completely resuspend the beads Place the tube on the magnetic separator for 1 minute Repeat wash Remove supernatant and resuspend beads in one bead volume of HT96 ChIP buffer Dilute antibody with HT96 ChIP buffer so that the concentration for each antibody is appropriate for HT96 ChIP 20 ng uL for most antibodies and add 100 uL or more of antibody to a standard 96 well plate not provided The amount of antibody used per ChIP must be empirically determined In general 1 10 ug of purified antibodies is generally sufficient for standard immunoprecipitations For Magna ChIP M HT96 3 uL of the Anti Trimethyl Histone H3 Lys4 control antibody CS207358 is sufficient for 100 000 cells and as little as 1 uL can be used for fewer cells The amount of antibody used per reaction may require optimization and in general lower amounts of antibody are appropriate for Magna ChIP HT96 reactions We recommend that you perform a negative control ChIP using normal IgG or no antibody Itis also a good idea to include replicates in your experiment Add diluted beads to a reservoir Place antibody plate magnetic separator and beads in Freedom EVO robotic workstation Place ChIP plate on top of magnetic separator Transfer 50 uL diluted antibodies and 50 uL beads to the ChIP plate using TECAN Cover and gently shake the plate to resuspend magnetic particles and place the plate on a
35. tes Optional At the end of the incubation homogenize the cell suspension 10 times in a Dounce homogenizer to facilitate the release of the nuclei Spin the cell suspension at 800 x g at 4 C for 5 minutes In the interim prepare 0 5 mL of HT96 ChIP Buffer containing 2 5 uL of 200X Protease Inhibitor Cocktail IIl for each microcentrifuge tube as before Step 1 Remove supernatant Resuspend cell pellets in HT96 ChIP Buffer from Step 7 For every 1 x 10 HeLa cells 0 5 mL of HT96 ChIP Buffer is recommended when using this protocol It is recommended that cell concentration is less than 2 x 10 cells mL as the ratio of lysis buffer to cell density is important for reliable cell lysis If optimal conditions for sonication have already been determined proceed to Section C Otherwise see Appendix A 10 C Sonication of Isolated Chromatin to Shear DNA Important Optimal conditions need to be determined to shear crosslinked DNA to 200 1000 base pairs in length See Appendix A for a typical optimization protocol Once shearing conditions have been optimized proceed with the steps below 1 7 D If desired remove 5 uL of cell lysate from Section B Step 7 for agarose gel analysis of unsheared DNA Sonicate cell lysate on wet ice ice water mixture The efficiency of sonication depends upon cell type cell equivalents and instrumentation When possible consult your instrument manufacturer s guidelines for inst
36. the solution Incubate at room temperature for 10 minutes Agitation of cells is not necessary Performing crosslinking in low serum conditions with culture media or PBS is optional as an optimization parameter to improve crosslinking efficiency Crosslinking time can be increased but may result in higher non specific association of DNAs with the ChIP antibody of interest During the ten minute incubation prepare 1X protease inhibitor in PBS Add 2 mL of ice cold 1X PBS to a separate tube for every dish and add 10 uL of the 200X Protease Inhibitor Cocktail Ill Store on ice Add 2 mL of 10X glycine to each dish to quench excess formaldehyde Swirl to mix and incubate at room temperature for 5 minutes Place dishes on ice Aspirate medium removing as much medium as possible being careful not to disturb the cells If you are using suspension cells spin down cells at 8000 x g for 5 minutes Add 10 mL of cold 1X PBS to wash cells Remove 1X PBS 12 13 14 15 Remove 1X PBS and repeat wash Add 2 mL of 1X Protease Inhibitor Cocktail III in PBS prepared in Step 6 Scrape cells from each dish into a separate microcentrifuge tube Spin at 800 x g at 4 C for 5 minutes to pellet cells ll Fresh tissue Isolate non fixed fresh tissue as desired Use a razor blade to cut a pea size piece of tissue into small pieces typically 1 mm or smaller to improve crosslink efficiency Alternatively a plug of tissue from cryosectioned non fo
37. tin and ChIP reagent requirements If you are inexperienced in the methodology of ChIP or unsure of the performance of your antibody in ChIP you may consider conducting a classical ChIP experiment using a kit such as the EZ Magna ChIP kit Cat No 17 10085 Overview of Magna ChIP HT96 Workflow Cells Tissues In vivo cross linking Lysis Isolation of chromatin cultured cells or tissues Sonication to shear chromatin 96 well plate immunoprecipitation 10 000 100 000 cells per reaction Manual or automated processing Reversal of cross links DNA purification optional v i Amplification bi Detection amp 1000 s Quantitative PCR ol e Promoter microarray 0 10 20 30 40 50 i s e Sequencing Detailed Protocol Chromatin Immunoprecipitation Please Read Entire Protocol First A In Vivo Crosslinking of Proteins to DNA l Cultured cells 1 11 If necessary stimulate or treat adherent mammalian cells at 80 to 90 confluence in a 150 mm culture dish containing 20 mL of growth media Include one extra plate of cells to be used solely for estimation of cell number For HeLa cells this is approximately 1 x 10 cells This will generate a preparation of chromatin sufficient for 100 separate immunoprecipitations when using 1 X 10 cell equivalents reaction Use the same amount of buffer when making chromatin from fewer cells The volume of
38. tion with the Magna ChIP HT96 kit is provided For your specific genomic region of interest a ChIP validated antibody demonstrated to enrich for your target region is required Millipore offers a wide selection of highly specific antibodies demonstrated to work in ChIP ChIP qualified antibodies as well as a collection of rigorously validated antibodies and control primer sets known as ChIPAb kits see page 6 or visit www millipore com epigenetics to search a complete list of targets Kit Components The Magna ChIP HT96 kit provides sufficient reagents for 24 individual chromatin preparation reactions and 96 chromatin immunoprecipitation reactions The EZ Magna ChIP HT96 kit includes these reagents plus positive and negative control antibodies and a human genome specific primer set for qPCR analysis For primers that can be used for non human cell lines or tissues visit www millipore com Magna ChIP HT96 High Throughput Chromatin Immunoprecipitation Kit Contents Kit Configurations Magna ChIP HT96 EZ Magna ChIP HT96 Cat No 17 10077 Cat No 17 10078 MAGNAO022 2C to 8C MAGNAO0022 2 to 8C MAGNAO023 20C MAGNAO0024 20C MAGNAO022 2 to 8 Component Catalog Quantity HT96 Nuclei Isolation Buffer CS207317 15 mL 10X Glycine CS207294 60 mL 10X PBS CS207282 60 mL HT96 ChIP Buffer Sonication ChIP Wash CS207283 80 mL Magna ChIP Protein A G Magnetic B
39. ute chromatin in a microcentrifuge tube and store at 20 C this will be used as 5 input t he following day Step 17 In order to get a useful standard curve during qPCR analysis the input should contain chromatin that is equivalent of 50 000 or more cells Otherwise use 5 uL of undiluted chromatin and adjust the percentage accordingly Spin down the plate briefly place the plate on the magnetic separator for 1 minute Remove supernatant then add 100 uL dilute chromatin to each well Cover and gently shake the plate to resuspend magnetic particles and place the plate on a rotating platform at 50 100 rpm overnight at 4C Ensure that the top of the plate is dry before applying the plastic seal seal tightly Spin down the plate at 800 x g for 1 minute then place the plate on the magnetic separator for 1 minute Remove supernatant being careful not to disturb the beads Add 100 uL cold HT96 ChIP buffer remove the plate from the magnetic separator and mix the beads by gently pipetting several times Place the plate on the magnetic separator for 1 minute then remove the supernatant Repeat wash with cold HT96 ChIP buffer twice as described in Step 13 Wash with HT96 Low Stringency IP Wash Buffer as before Step 13 12 16 17 18 19 20 Resuspend the beads in 100 uL HT96 Low Stringency Buffer then transfer to 96 well Thermal Plate Place the 96 well Thermal Plate on the magnetic separator for 1 minute then rem
40. y Figure 2 Sonicated chromatin prepared from 100 000 untreated HeLa cells A or 10 000 untreated HeLa cells B was subjected to chromatin immunoprecipitation using 1 ug of purified IgG mouse IgG 12 371B Rabbit IgG 12 370 or specific antibodies H3K4Me3 17 614 H3K9Ac 17 658 Phospho CREB 17 10131 RNA Pol Il 17 620 and the Magna ChIP HT96 Kit using a Freedom EVO robotic workstation Immunoprecipitation of antibody associated DNA fragments was verified by qPCR using control primers flanking the human GAPDH promoter region Standard deviation of qPCR triplicates is shown and results reflect analysis of 2 uL out of 50 uL total DNA per qPCR reaction Technical Replicate Analysis Using Automated ChIP Standard Deviation of Technical Replicates N 4 Percent of Input 0 02 H3K4Me3 Control IgG Replicate Analysis N 4 Using 100 000 Cell Equivalents and Automated ChIP 1 0 q 0 8 a 3 e 0 6 El Test 1 5 B Test 2 t n Test 3 8 04 n Test 4 o a 0 2 4 0 0 H3K4Me3 Control IgG pCREB Control IgG R S133 M Figure 3 Sonicated chromatin prepared from 100 000 untreated HeLa cells was subjected to chromatin immunoprecipitation using 1 ug of purified IgG mouse IgG 12 371B Rabbit IgG 12 370 or specific antibodies H3K4Me3 17 614 Phospho CREB 17 10131 and the Magna ChIP HT96 Kit using a Freedom EVO robotic workstation Immunoprecipitation of antibody associated DNA frag
Download Pdf Manuals
Related Search