Affymetrix Chromatin Immunoprecipitation Assay
Contents
1. Material Supplier Part Number Instruments Rotating Benchtop Platforms Various Branson Sonifier S 450D Branson Ultrasonics 101 063 590 Double Step Micro Tip Assembly Branson Ultrasonics 101 063 212 NanoDrop ND 1000 Nanodrop Technologies ND 1000 GeneChip Hybridization Oven 640 Affymetrix 8001318 Eppendorf Centrifuge Eppendorf 5417C Refrigerated Centrifuge with swing bucket rotor Various Tube Strip Picofuge Stratagene 400540 GeneChip Fluidics Station 450 or 400 Affymetrix 00 0079 GeneChip Scanner 3000 7G Affymetrix 00 0073 GeneChip Autoloader Optional Affymetrix 90 0351 ABI GeneAmp PCR System 9700 Applied Biosystems N A Bioanalyzer 2100 Agilent G2940CA Heating Block VWR 13259 030 Pipette for 0 1 to 2 uL Rainin L 2 Pipette for 2 to 20 uL Rainin L 20 Pipette for 20 to 200 uL Rainin L 200 Pipette for 100 to 1 000 uL Rainin L 1000 Or equivalent instrument supplies Chapter 2 Chro Chapter 2 15 Procedure A Prepare Cells Grow enough cells for the number of immunoprecipitation IP reactions to be performed usually 5 x 107 cells per IP for suspension cells depending on IP efficiency Prepare enough cells for two IP reactions An antibody minus Ab or mock IP or non specific IgG is recommended as a negative control using the same number of cells as the IP condition The Ab target would be treated identically to the experimental sample to serve as the Control group2. Affymetrix Chromatin Immunoprecipitation Assay Protocol P N 702238 Rev 3 For research use only Not for use in diagnostic procedures Trademarks a Affymetrix GeneChip AMC HuSNP GenFlex Flying Objective CustomExpress CustomSeq NetAffx Tools To Take You As Far As Your Vision The Way Ahead Powered by Affymetrix and GeneChip compatible are trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered by one or more of the following patents and or sold under license from Oxford Gene Technology U S Patent Nos 5 445 934 5 700 637 5 744 305 5 945 334 6 054 270
3. 7 Table 1 1 Continued Materials Required Material Source Part Number PCR Amplification Sequenase Version 2 0 DNA Polymerase USB 70775Y Primer A 200 M GTTTCCCAGTCACGGTC N Various HPLC purified Primer B 100 uM GTTTCCCAGTCACGGTC Various HPLC purified Taq Polymerase 5 U uL Various 10X PCR Buffer Various dATP 100 mM Various dCTP 100 mM Various dGTP 100 mM Various dTTP 100 mM Various dUTP 100 mM Various BSA 20 mg mL Various DTT 0 1M Various Wash Buffer Tris HCl Various EDTA Various SDS 100g Sigma Aldrich 71725 NaCl Various Deoxycholate sodium salt 100 g Sigma Aldrich D6750 MgCl 1M Various CaCl 1M Sigma Aldrich 21115 Fragmentation and Labeling GeneChip WT Double Stranded DNA Terminal Labeling Kit Affymetrix 900812 30 Rxn 8 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table 1 1 Continued Materials Required Material Source Part Number DNA Cleanup GeneChip Sample Cleanup Module 30 Rxn Affymetrix 900371 ERC Buffer 85 mL OIAGEN 1018144 Hybridization Stain and Wash GeneChip Hybridization Wash and Stain Kit Affymetrix 900720 Control Oligonucleotide B2 3nM Affymetrix 900301 NOTE The GeneChip Sample Cleanup Module includes 20 mL of cDNA Binding Buffer In order to process 30 samples following the Affymetrix Chromatin Immunoprecipitation Assay Protocol addition
4. provided in the GeneChip Sample Cleanup Module eluting twice with 20 pL of Elution Buffer 10 Measure DNA using a NanoDrop or other UV vis spectrophotometer Normally greater than 9 pg of amplified DNA is obtained from each reaction Maintenance of IP enrichment post amplification is crucial in obtaining good array results QPCR should be performed to post amplified samples to ensure that differences between the IP and Ab samples are maintained Primer sets can be designed for DNA regions that are known to be specifically immunoprecipitated using the antibody of interest chapter 2 Chromatin Immunoprecipitation Assay 29 Procedure Fragment Amplified Targets 1 Fragment the samples using the appropriate table below depending on what array type the target will be hybridized to Table 2 5 Fragmentation Mix for single arrays e g Human Promoter 1 0R Array Double Stranded DNA 7 5 ug 10X cDNA Fragmentation Buffer 4 8 uL UDG 10 U uL 1 5 uL APE 1 100 U uL 2 25 uL Nuclease free Water up to 48 uL Total Volume 48 0 uL Available in GeneChip WT Double Stranded DNA Terminal Labeling Kit P N 900812 Table 2 6 Fragmentation Mix for multi array sets e g Human Tiling 2 0R Array Set Double Stranded DNA 9 ug 10X cDNA Fragmentation Buffer 4 8 uL UDG 10 U uL 1 5 uL APE 1 100 U uL 2 25 uL Nuclease free Water up to 48 uL Total Volume 48 0 uL Available in Gen
5. Protocol Optimization This protocol has been developed for use with GeneChip Tiling Arrays Exact protocol conditions will require optimization by each user due to the variability inherent in e Experimental Biology cell types proteins of interest antibodies Assay conditions DNA fragmentation PCR conditions To ensure success with this protocol it is critical that users optimize the following steps in the ChIP protocol prior to performing microarray hybridizations Additional information on the optimization steps are available throughout this protocol 1 Sonication conditions of fixed cells Some cells are resistant to sonication treatment Micrococcal nuclease treatment may improve DNA shearing for some cell lines Antibody Qualification Antibodies should be qualified for use with chromatin immunoprecipitation experiments ChIP qualification information is available from www chiponchip org or directly from antibody vendors Antibody Titration Antibody affinities and avidities can vary so the amount of antibody may need to be titrated to achieve optimal sample enrichment PCR amplification of immunoprecipitated DNA The optimal number of PCR cycles may require optimization to avoid saturation and ensure that the IP enrichment is maintained QPCR Positive Control A QPCR control is recommended to test ChIP conditions This test requires a known protein binding to a known DNA sequence After performing ChIP
6. Volume Final Concentration 2X Stain Buffer see page 63 300 uL 1X 50 mg mL BSA 24 pL 2 mg mL 1 mg mL Streptavidin Phycoerythrin SAPE 6 uL 10 ug mL Molecular Biology Grade Water 270 uL _ Total Volume 600 uL Mix well The 600 pL of SAPE Solution Mix will be used for the 1 and 3 stain If using the Fluidics Station 400 after the first stain is done save the tube with the SAPE stain solution Reuse the saved tube for the third stain 66 Affymetrix Chromatin Immunoprecipitation Assay Protocol Antibody Solution Table B 6 Antibody Solution Mix Components Volume Final Concentration 2X Stain Buffer 300 0 uL 1X 50 mg mL BSA 24 0 uL 2 mg mL 10 mg mL Goat IgG Stock 6 0 uL 0 1 mg mL 0 5 mg mL biotinylated 3 6 uL 3 ug mL antibody Molecular Biology Grade Water 266 4 uL _ Total Volume 600 uL Mix well The 600 pL of Antibody Solution Mix will be used for the 29 Stain Array Holding Buffer To prepare the Array Holding Buffer refer to Table B 2 on page 63 Appendix C Contact Information Appendix C 69 Contact Information Affymetrix Inc 3420 Central Expressway Santa Clara CA 95051 USA E mail support affymetrix com Tel 1 888 362 2447 1 888 DNA CHIP Fax 1 408 731 5441 Affymetrix UK Ltd Voyager Mercury Park Wycombe Lane Wooburn Green High Wycombe HP10 OHH United Kingdom E mail supporteurope affymetrix com UK and Others T
7. in the downstream two sample analysis Use 0 5 2 x 10 cells per IP For example grow 200 mL of 1 x 106 cells mL for a total of 2 x 10 cells Procedure B Fix Cells Lyse and Sonicate Whole Cell Extracts NOTE ADHERENT CELLS NOTE DAY 1 Centrifugation steps involving cells are best performed with a swing bucket type rotor End users may optimize the sequence of fixing and harvesting cells to minimize the degree to which cell physiology is disrupted 1 Add formaldehyde to the culture flask to a final concentration of 1 and incubate in a fume hood for 10 minutes Add 1 20 volume of 2 5 M glycine and incubate at room temperature RT for 5 minutes with gentle mixing Pour off formaldehyde media into an appropriate waste container and add enough ice cold 1X PBS to cover the bottom of the flask to wash cells Pour off PBS into a formaldehyde waste container and add enough PBS to cover bottom of flask 16 Affymetrix Chromatin Immunoprecipitation Assay Protocol SUSPENSION CELLS 1 WASH CELL PELLET 1 Using a cell scraper scrape off cells to re suspend and check flask with microscope to ensure that most cells are re suspended From here go to Step 1 of the Wash Cell Pellet section below Fix cells by adding formaldehyde to a final concentration of 1 add 5 5 mL of 37 formaldehyde to 200 mL of culture medium Incubate at room temperature RT in fume hood for 10 minutes gently swi
8. it on at least 10 minutes prior to use If probe array was stored at 4 C warm to room temperature before scanning Refer to the GCOS online help and the appropriate scanner user s manual for more information on scanning The scanner uses a laser and is equipped with a safety interlock system Defeating the interlock system may result in exposure to hazardous laser light You must have read and be familiar with the operation of the scanner before attempting to scan a probe array Please refer to the the GeneChip Scanner 3000 Quick Reference Card or user s manual HANDLING THE PROBE ARRAY IMPORTANT Li Before you scan the probe array follow the directions in this section on handling the probe array If necessary clean the glass surface of the probe array with a non abrasive towel or tissue before scanning Do not use alcohol to clean glass Before scanning the probe array cartridge apply Tough Spots label dots to each of the two septa on the probe array cartridge to prevent the leaking of fluids from the cartridge during scanning Apply the spots just before scanning 1 On the back of the probe array cartridge clean excess fluid from around septa 2 Carefully apply one Tough Spots to each of the two septa Press to ensure that the spots remain flat If the Tough Spots do not apply smoothly that is if you observe bumps bubbles tears or curled edges do not attempt to smooth out the spot Remove the spot an
9. with an antibody to the known protein QPCR is used to verify that the known DNA binding elements are enriched in experimental vs negative control samples This QPCR test can also be used to ensure that the enrichment of experimental samples vs control samples is maintained after IP column clean up chapter 1 Overview Fix cells to E crosslink DNA to protein Sonicate to lyse O cells and shear D lt P lt n TEL lt lt chromatin L Day 1 Take small aliquot to decrosslink and check lt lt v sonication efficiency Immunoprecipitate Er A main sample with DaT Dan roere lt lt selected antibody Couple to la ye i Protein A beads and wash to purify IP d DNA L Decrosslink and A proteinase treat Day 2 IP d DNA GeneChIP Genus Sample Cleanup IP d DNA T DRE Module a Random Adapter ji Primer Adapter Sequenase dNTPs A Linear E 5111113 Amplification q 311111111115 y Adapter Primer dNTP dUTP we Taq polymerase 5111113 O II II IT IT 5 PCR amplify and incorporate dUTP y 5 11U1113 STIUITITUIIT3 3 III Ul 5 SI II TUI 1U15 Uracil DNA Glycosylase L Day3 Fragmentation yO APE 1 I 11 111 L L Fragment and p A label ampli
10. 00 pL TE 10 mM Tris HCl pH 8 1 mM EDTA Incubate on rotating platform at RT for 1 minute Centrifuge at 2 000 rpm at RT and discard flow through Repeat steps 17 through 19 Transfer the spin X column with beads to a dolphin nose tube Add 200 pL Elution Buffer to the column Incubate at 65 C for 30 minutes Centrifuge at 3 000 rpm at RT for 2 minutes Add 200 pL Elution Buffer to the column Centrifuge at 3 000 rpm at RT for 2 minutes This 400 pL eluted sample is the enriched or IP d sample Procedure F Reverse Crosslinks 1 2 Add 5 pL Proteinase K 20mg mL per 100 pL of negative control or IP sample mix well 20 pL for 400 pL of eluted sample Incubate in incubator at 65 C overnight chapter 2 Chromatin Immunoprecipitation Assay 23 DAY 3 Procedure G Cleanup De crosslinked Samples 1 Clean up samples using Affymetrix cDNA cleanup columns Elute twice with 20 pL Elution Buffer Appendix A page 55 Total elution volume recovered is 38 pL 2 IP efficiency can be checked at this stage in the protocol using polymerase chain reaction PCR and designing primer sets against regions that are known to be bound by the protein of interest and immunoprecipitated using the antibody being investigated A significant increase or enrichment for the specific target should be observed for the IP condition compared to the Ab control Using quantitative real time PCR Affymetrix has routinely obt
11. 4 Wash and Stain the Probe Array on Fluidics Station 450 45 Shut Down the Fluidics Station 48 Scanning 49 SCAN 51 Handling the Probe Array 51 Scanning the Probe Array 53 Cleanup of Double Stranded DNA 55 CLEANUP OF DOUBLE STRANDED DNA 57 APPENDIX B APPENDIX C contents Buffers and Solutions Required for Array Hybridization Washing and Staining 59 BUFFERS AND SOLUTIONS REQUIRED FOR ARRAY HYBRIDIZATION WASHING AND STAINING 61 PREPARING THE STAINING REAGENTS 65 Contact Information 67 CONTACT INFORMATION 69 iv Affymetrix Chromatin Immunoprecipitation Assay Protocol Chapter 1 Overview Chapter 1 Introduction The Affymetrix Chromatin Immunoprecipitation ChIP Assay is designed to generate double stranded labeled DNA targets that identify sites of protein DNA interactions or chromatin modifications on a genome wide scale This assay has been designed specifically for use with Affymetrix GeneChip Tiling Arrays for ChIP on chip studies in order to study transcription factor binding sites histone protein modifications and other chromatin protein interactions ChIP experiments can be used as a powerful tool to complement RNA transcription studies because they enable researchers to study the DNA protein interactions that regulate gene expression Following the protocol cells are first fixed with formaldehyde to crosslink DNA to any associated proteins The cells are then lysed and DNA is s
12. 6 140 044 6 261 776 6 291 183 6 346 413 6 399 365 6 420 169 6 551 817 6 610 482 6 733 977 and EP 619 321 373 203 and other U S or foreign patents Copyright 2005 2006 Affymetrix Inc All rights reserved CHAPTER 1 CHAPTER 2 Contents Overview 1 INTRODUCTION 3 CHROMATIN IMMUNOPRECIPITATION ASSAY PROTOCOL OPTIMIZATION 4 MATERIALS 6 Buffers 9 Miscellaneous Reagents and Supplies 11 Chromatin Immunoprecipitation Assay 13 PROCEDURE A PREPARE CELLS 15 PROCEDURE B FIX CELLS LYSE AND SONICATE WHOLE CELL EXTRACTS 15 Adherent Cells 15 Suspension Cells 16 Wash Cell Pellet 16 PROCEDURE C CHECK SONICATION EFFICIENCY 18 PROCEDURE D INCUBATE WITH SPECIFIC ANTIBODY 20 PROCEDURE E IMMUNOPRECIPITATE AND WASH 21 PROCEDURE F REVERSE CROSSLINKS 22 PROCEDURE G CLEANUP DE CROSSLINKED SAMPLES 23 PROCEDURE H PCR AMPLIFY IMMUNOPRECIPITATED DNA TARGETS 23 PROCEDURE FRAGMENT AMPLIFIED TARGETS 29 PROCEDURE J LABEL FRAGMENTED DOUBLE STRANDED DNA 31 CHAPTER 3 CHAPTER 4 CHAPTER 5 APPENDIX A ii Affymetrix Chromatin Immunoprecipitation Assay Protocol Hybridization and Array Processing 33 PROCEDURE A HYBRIDIZE LABELED TARGET ON THE ARRAYS 35 Array Washing and Staining 39 PROCEDURE A ENTER EXPERIMENT INFORMATION 41 PROCEDURE B PREPARE THE FLUIDICS STATION 42 Set Up the Fluidics Station 42 Prime the Fluidics Station 42 PROCEDURE C WASH AND STAIN PROBE ARRAYS 43 Fluidics Protocols 4
13. 7889 DMSO Sigma Aldrich D5879 Surfact Amps 20 Tween 20 10 Pierce 28320 PBS pH 7 2 Invitrogen 20012 027 20X SSPE 3M NaCl 0 2M NaH PO 0 02M EDTA Cambrex 51214 Tough Spots Label Dots USA Scientific 9185 0000 Available in the GeneChip Hybridization Wash and Stain Kit P N 900720 62 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table B 2 Buffers Required to be Prepared 12X MES Stock Buffer 1 22M MES 0 89M Na For 1 000 mL 64 61g of MES hydrate 193 3g of MES Sodium Salt 800 mL of Molecular Biology Grade water Mix and adjust volume to 1 000 mL The pH should be between 6 5 and 6 7 Filter through a 0 2 um filter IMPORTANT Do not autoclave Store at 2 C to 8 C and shield from light Discard solution if yellow 2X Hybridization Buffer Final 1X concentration 100 mM MES 1M Nat 20 mM EDTA 0 01 Tween 20 For 50 mL 8 3 mL of 12X MES Stock Buffer 17 7 mL of 5M NaCl 4 0 mL of 0 5M EDTA 0 1 mL of 10 Tween 20 19 9 mL of water IMPORTANT Store at 2 C to 8 C and shield from light Wash Buffer A Non Stringent Wash Buffer included in P N 900720 6X SSPE 0 01 Tween 20 For 1 000 mL 300 mL of 20X SSPE 1 0 mL of 10 Tween 20 699 mL of water Filter through a 0 2 um filter appendix B Buffers and Solutions Required for Array Hybridization Washing and Staining 63 Table B 2 Continued Buffers Required to be Prepared Wash Buff
14. 9 minutes 37 C for 8 minutes 95 C for 4 minutes Snap cool on ice 10 C hold Add 1 0 pL of 1 3U pL SequenaseTM to each sample 10 C for 5 minutes Ramp from 10 C to 37 C over 9 minutes 37 C for 8 minutes Repeat from J to P for 2 more cycles 4 C hold For each IP purify with Microspin S 300 HR GE Healthcare columns 2 columns per reaction as follows A Add 20 pL of 10 mM TE pH 8 0 to each reaction B Spin 2 columns A amp B at 3 000 rpm for 1 minute discard flow through Sp VO Z2 ZT AS C Transfer reaction volume 43 pL to column A while equilibrating column B with 300 pL of 10 mM Tris pH 8 0 D Spin both columns at 3 000 rpm for 1 minute keep flow through from column A sample and discard flow through of column B Tris buffer E Transfer flow through of column A to column B with new collection tube F Spin at 3 000 rpm for 2 minutes G Collect 56 pL of first round purified DNA per reaction 26 Affymetrix Chromatin Immunoprecipitation Assay Protocol 5 Prepare INTP dUTP mix Prior to proceeding with the PCR amplification of immunoprecipitated DNA targets prepare a dNTP mixture containing dUTP at the concentrations indicated below Please note that this dNTP dUTP mixture is only required for the PCR amplification reaction outlined in Table 2 4 and not in the Sequenase reaction setup in Table 2 3 dCTP 10 mM dATP 10 mM dGTP 10 mM dTTP 8
15. ASH AND STAIN THE PROBE ARRAY ON FLUIDICS STATION 450 NOTE If a Fluidics Station 450 instrument is unavailable proceed with washing and staining with the appropriate FS400 fluidics protocol Add Holding Buffer to the cartridge manually prior to scanning 1 In the Fluidics Station dialog box on the workstation select the correct experiment name from the drop down Experiment list The Probe Array Type appears automatically 2 In the Protocol drop down list select FS450_0001 or FS450_0002 to control the washing and staining of the probe array 3 Choose Run in the Fluidics Station dialog box to begin the washing and staining Follow the instructions in the LCD window on the fluidics station If you are unfamiliar with inserting and removing probe arrays from the fluidics station modules please refer to the appropriate Fluidics Station User s Guide or Quick Reference Card PIN 08 0093 for the FS 450 250 fluidics station 4 Insert the appropriate probe array into the designated module of the fluidics station while the cartridge lever is in the down or eject position When finished verify that the cartridge lever is returned to the up or engaged position 46 Affymetrix Chromatin Immunoprecipitation Assay Protocol Remove any microcentrifuge vials remaining in the sample holders of the fluidics station module s being used Follow the instructions on the LCD window Place the following three vials the microce
16. Labeled DNA Target 60 uL 7 5 ug Control Oligonucleotide B2 3 3 uL 50 pM Herring Sperm DNA 10 mg mL 2 0 uL 0 1 mg mL Acetylated BSA 50 mg mL 2 0 uL 0 5 mg mL 2X Hybridization Buffer 100 uL 1X DMSO 14 uL 7 RNase free Water up to 200 0 uL Total Volume 200 0 uL Table B 4 Hybridization Cocktail for use with serial hybridizations e g GeneChip Human Tiling 2 0R Array Set and GeneChip Mouse Tiling 2 0R Array Set if not using the GeneChip Hybridization Wash and Stain Kit Component Volume in Final Concentration 1 Reaction or Amount Fragmented and Labeled DNA Target 60 uL 9 0 ug Control Oligonucleotide B2 4 0 uL 50 pM Herring Sperm DNA 10 mg mL 2 4 uL 0 1 mg mL Acetylated BSA 50 mg mL 2 4 uL 0 5 mg mL 2X Hybridization Buffer 120 uL 1X DMSO 16 8 uL 7 RNase free Water up to 240 0 uL Total Volume 240 0 uL appendix B Buffers and Solutions Required for Array Hybridization Washing and Staining 65 Preparing the Staining Reagents Prepare the following reagents Volumes given are sufficient for one probe array SAPE Stain Solution Streptavidin Phycoerythrin SAPE should be stored in the dark at 4 C either foil wrapped or kept in an amber tube Remove SAPE from the refrigerator and tap the tube to mix well before preparing stain solution Do not freeze SAPE Always prepare the SAPE stain solution fresh on the day of use Table B 5 SAPE Solution Mix Components
17. ained gt 8 fold enrichment for IP samples compared to the Ab samples Procedure H PCR Amplify Immunoprecipitated DNA Targets NOTE Dilute Sequenase stock with Sequenase Dilution Buffer included with enzyme to 1 3 U uL Four microliters of this 1 3 U uL working stock will be needed for each sample being amplified 1 Use 10 pL of IP d or negative control sample for initial round of linear amplification 24 Affymetrix Chromatin Immunoprecipitation Assay Protocol 2 Set up first round reaction Set up 1 reaction for single array products e g Human Promoter 1 0R Array Setup 3 reactions for multi array sets e g Human Tiling 2 0R Array Set Table 2 2 Purified DNA 10 uL 5X Sequenase Reaction Buffer 4 pL Primer A 200 uM 4uL Total Volume 18 uL included with enzyme t Primer A GTTTCCCAGTCACGGTCIN s HPLC purified 3 Cycle conditions Random priming A 95 C for 4 minutes B Snap cool samples on ice C 10 C hold D Prepare first cocktail Table 2 3 First Cocktail Component Volume for 1 Rxn 20 mg mL BSA 0 1 uL 0 1 M DTT 1 uL 25 mM dNTPs 0 5 uL Diluted Sequenase 1 10 from 13 U uL stock 1 uL Total Volume 2 6 uL E Add 2 6 pL per sample F Mix well by pipetting and put the sample back in thermocycler block chapter 2 Chromatin Immunoprecipitation Assay 25 G 10 C for 5 minutes H Ramp from 10 C to 37 C over
18. al cDNA Binding Buffer is required This buffer should be purchased directly from QIAGEN When purchasing the cDNA Binding Buffer from QIAGEN please order ERC Buffer part number 1018144 chapter 1 Overview 9 BUFFERS Table 1 2 Buffers Lysis Buffer Store at 4 C 10 mM Tris HCI made from stock 1M Tris HCI pH 7 5 10 mM NaCl 3 mM MgCl 0 5 IGEPAL 1 mM PMSF add fresh Pre IP Dilution Buffer Store at RT 10 mM Tris HCl made from stock 1M Tris HCI pH 7 5 10 mM NaCl 3 mM MgCl 1 mM CaCl 4 IGEPAL 1 mM PMSF add fresh IP Dilution Buffer Store at RT without protease inhibitors 20 mM Tris HCI made from stock 1M Tris HCl pH 8 2mM EDTA 1 Triton X 100 150 mM NaCl Protease Inhibitor Stock add fresh Protease Inhibitor Stock Prepare a 25X stock by dissolving 1 protease inhibitor tablet in 2 mL of nuclease free water ChIP Wash 1 Store at RT 20 mM Tris HCI made from stock 1M Tris HCl pH 8 2mM EDTA 1 Triton X 100 150 mM NaCl 1 mM PMSF add fresh 10 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table 1 2 Continued Buffers ChIP Wash 2 Store at RT 20 mM Tris HCI made from stock 1M Tris HCI pH 8 2 mM EDTA 1 Triton X 100 0 1 SDS 500 mM NaCl 1 mM PMSF add fresh ChIP Wash 3 Store at RT 10 mM Tris HCI made from stock 1M Tris HCI pH 8 1 mM EDTA 0 25M LiCl 0 5 IGEPAL 0 5 Deoxycholate sodium sa
19. and then Experiment Name PROJECT v SAMPLE y EXPERIMENT 42 Affymetrix Chromatin Immunoprecipitation Assay Protocol Procedure B Prepare the Fluidics Station The GeneChip Fluidics Station 450 250 or 400 is used to wash and stain GeneChip Tiling Arrays It is operated using GCOS Use the GeneChip Hybridization Wash and Stain Kit P N 900720 or prepare buffers as indicated in Appendix B SET UP THE FLUIDICS STATION 1 Turn on the Fluidics Station using the toggle switch on the lower left side of the machine 2 Select Run Fluidics from the menu bar The Fluidics Station dialog box appears with a drop down list for selecting the experiment name for each of the fluidics station modules A second drop down list is accessed for choosing the Protocol for each of the fluidics station modules NOTE Refer to the Fluidics Station User s Guide for instructions on connecting and addressing multiple fluidics stations PRIME THE FLUIDICS STATION Priming ensures that the lines of the GeneChip Fluidics Station are filled with the appropriate buffers and the Fluidics Station is ready for running fluidics station protocols Priming should be done when the Fluidics Station is first started when wash solutions are changed before washing if a shutdown has been performed e if the LCD window instructs the user to prime 1 To prime the fluidics station select Protocol in t
20. anded DNA Labeling Mix Component Volume in 1 Rxn 5x TdT Buffer 12 uL TdT 2uL DNA Labeling Reagent 5 mM 1 UL Total Volume 15 uL Available in the GeneChip WT Double Stranded DNA Terminal Labeling Kit P N 900812 2 Add 15 pL of the Double Stranded DNA Labeling Mix to the DNA samples flick mix and spin them down 3 Incubate the reactions at e 37 C for 60 minutes e 70 C for 10 minutes e 4 C for at least 2 minutes 4 Remove 2 pL of each sample for gel shift analysis refer to the GeneChip Whole Transcript WT Sense Target Labeling Assay Manual 32 Affymetrix Chromatin Immunoprecipitation Assay Protocol Chapter 4 Hybridization and Atay Pois Chapter 3 35 Procedure A Hybridize Labeled Target on the Arrays This Procedure requires the use of the GeneChip Hybridization Wash and Stain Kit P N 900720 Alternatively users may prepare their own hybridization mix using Table B 2 and either Table B 3 or Table B 4 in Appendix B 1 Prepare the Hybridization Cocktail in a 1 5 mL RNase free microfuge tube as shown in Table 3 1 and Table 3 2 below depending on what array type the target will be hybridized to Table 3 1 Hybridization Cocktail for single tiling arrays e g GeneChip Human Promoter 1 0R Array Component Volume in 1 Final Concentration Rxn or Amount Fragmented and Labeled DNA Target 60 0 uL 7 5 ug Control Oligonucleotide B2 3 3 uL 50
21. ating platform at 4 C overnight or for at least 3 hours at RT chapter 2 Chromatin Immunoprecipitation Assay 21 DAY 2 Procedure E Immunoprecipitate and Wash 1 Pre equilibrate protein A SepharoseTM beads by adding 1 mL IP Dilution Buffer and 200 pL beads for each IP d sample Centrifuge 2 000 rpm 2 minutes at 4 C Discard around 800 pL supernatant save 400 pL of beads in buffer at the bottom of the tube 3 Transfer 400 pL beads to each sample 4 Add PMSF to each tube sample final concentration 1mM PMSF 10 11 12 13 14 15 16 in final volume Incubate on rotating platform at RT for 1 to 3 hours Centrifuge at 2 000 rpm at 4 C for 4 minutes and then discard supernatant Resuspend the pellet with 700 pL ChIP wash 1 containing 1 mM PMSF added fresh mix and transfer to spin X column Incubate on rotating platform at RT for 1 minute Centrifuge at 2 000 rpm at RT for 2 minutes and discard flow through Repeat steps 7 9 Wash the beads with 700 pL ChIP wash 2 containing 1 mM fresh PMSF Incubate on rotating platform at RT for 5 minutes Centrifuge at 2 000 rpm at RT and discard flow through Wash the beads with 700 pL ChIP wash 3 Incubate on rotating platform at RT for 5 minutes Centrifuge at 2 000 rpm at RT and discard flow through 22 Affymetrix Chromatin Immunoprecipitation Assay Protocol 17 18 19 20 21 22 23 24 25 26 Wash the beads with 7
22. cs Station on page 48 chapter 4 Array Washing and Staining 47 Table 4 4 If Bubbles are Present Return the probe array to the probe array holder Engage the washblock by gently pushing up on the cartridge lever to the engaged or closed position Follow the instructions on the LCD window The fluidics station will drain the probe array and then fill it with a fresh volume of Array Holding Buffer When finished the LCD window displays EJECT CARTRIDGE Again remove the probe array and inspect for bubbles If no bubbles are present it is ready to be scanned Proceed to Chapter 5 Scanning on page 49 If your attempt to fill the probe array without bubbles is unsuccessful manually drain the 1x Array Holding Buffer from the array using a micropipette and fill the array completely with a fresh aliquot of 1x Array Holding Buffer Inspectthe array and ensure that no bubbles are present Excessive washing will result in a loss of signal intensity 48 Affymetrix Chromatin Immunoprecipitation Assay Protocol SHUT DOWN THE FLUIDICS STATION IMPORTANT Li 1 After removing a probe array from the probe array holder the LCD window displays the message ENGAGE WASHBLOCK 2 If you are using the Fluidics Station 400 latch the probe array holder by gently pushing up until a light click is heard Engage the washblock by firmly pushing up on the cartridge lever to the ENGAGE position If you are using the Fluidics Statio
23. d apply a new spot See Figure 5 1 52 Affymetrix Chromatin Immunoprecipitation Assay Protocol Figure 5 1 Applying Tough Spots to the probe array cartridge 3 Insert the cartridge into the scanner and test the autofocus to ensure that the Tough Spots do not interfere with the focus If you observe a focus error message remove the spot and apply a new spot Ensure that the spots lie flat chapter 5 Scanning 53 SCANNING THE PROBE ARRAY 1 Select Run Scanner from the menu bar Alternatively click the Start Scan icon in the tool bar The Scanner dialog box appears with a drop down list of experiments that have not been run Select the experiment name that corresponds to the probe array to be scanned A previously run experiment can also be selected by using the Include Scanned Experiments option box After selecting this option previously scanned experiments appear in the drop down list Once the experiment has been selected click the Start button A dialog box prompts you to load an array into the scanner Open the sample door on the scanner and insert the probe array into the holder Do not force the probe array into the holder Close the sample door of the scanner Click OK in the Start Scanner dialog box The scanner begins scanning the probe array and acquiring data When Scan in Progress is selected from the View menu the probe array image appears on the screen as the scan prog
24. eChip WT Double Stranded DNA Terminal Labeling Kit P N 900812 30 Affymetrix Chromatin Immunoprecipitation Assay Protocol 2 Set up fragmentation mix according to either Table 2 5 or Table 2 6 Flick mix and spin down the tubes 3 Incubate the reactions at e 37 C for 1 hour e 93 C for 2 minutes 4 C for at least 2 minutes 4 Flick mix spin down the tubes and transfer 45 pL of the sample to a new tube 5 The remainder of the sample is to be used for fragmentation analysis using a Bioanalyzer or agarose gel Please see the Reagent Kit Guide that comes with the RNA 6000 LabChip Kit for instructions If not labeling the samples immediately store the fragmented DNA at 20 C Fluorescence Migration Time Figure 2 3 Bioanalyzer trace of fragmentation products following treatment of amplified ChIP targets with UDG and APE 1 Independently amplified Sp1 IP or Ab samples from HL 60 cells were fragmented according to the protocol and products were analyzed on an Agilent Bioanalyzer with the RNA 6000 Nano LabChip Kit Analyzing fragmented DNA on the RNA 6000 LabChip is recommended because it quickly assesses the degree and uniformity of the fragmented products chapter 2 Chromatin Immunoprecipitation Assay 31 Procedure J Label Fragmented Double Stranded DNA 1 Prepare the Double Stranded DNA Labeling Mix as described in Table 2 7 Table 2 7 Double Str
25. el 44 0 1628 552550 France Tel 0800919505 Germany Tel 01803001334 Fax 44 0 1628 552585 Affymetrix Japan K K Mita NN Bldg 16 Floor 4 1 23 Shiba Minato ku Tokyo 108 0014 Japan Tel 03 5730 8200 Fax 03 5730 8201 70 Affymetrix Chromatin Immunoprecipitation Assay Protocol
26. er B Stringent Wash Buffer included in P N 900720 100 mM MES 0 1M Na 0 01 Tween 20 For 1 000 mL 83 3 mL of 12X MES Stock Buffer 5 2 mL of 5M NaCl 1 0 mL of 10 Tween 20 910 5 mL of water Filter through a 0 2 um filter IMPORTANT Store at 2 C to 8 C and shield from light 2X Stain Buffer Final 1X concentration 100 mM MES 1M Nat 0 05 Tween 20 For 250 mL 41 7 mL of 12X MES Stock Buffer 92 5 mL of 5M NaCl 2 5 mL of 10 Tween 20 113 3 mL of water Filter through a 0 2 um filter IMPORTANT Store at 2 C to 8 C and shield from light 10 mg mL Goat IgG Stock Resuspend 50 mg in 5 mL of 150 mM NaCl Store at 4 C IMPORTANT If a larger volume of the 10 mg mL IgG stock is prepared aliquot and store at 20 C until use After the solution has been thawed it should be stored at 4 C Avoid additional freezing and thawing 1X Array Holding Buffer 100 mM MES 1M Nat 0 01 Tween 20 For 100 mL 8 3 mL of 12X MES Stock Buffer 18 5 mL of 5M NaCl 0 1 mL of 10 Tween 20 73 1 mL of water Store at 2 C to 8 C and shield from light 64 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table B 3 Hybridization Cocktail for single tiling arrays e g GeneChip Human Promoter 1 0R Array if not using the GeneChip Hybridization Wash and Stain Kit Component Volume in FinalConcentration 1 Reaction or Amount Fragmented and
27. f 15 mixes cycle with Wash Buffer B at 50 C 6 cycles of 15 mixes cycle with Wash Buffer B at 50 C Stain Stain the probe array for 10 minutes in SAPE solution at 35 C Stain the probe array for 5 minutes in SAPE solution Stain Cocktail 1 at 35 C Post Stain Wash 10 cycles of 4 mixes cycle with Wash Buffer A at 30 C 10 cycles of 4 mixes cycle with Wash Buffer A at 30 C Buffer A at 35 C The holding temperature is 25 C 2nd Stain Stain the probe array for 5 minutes in Stain the probe array for 5 minutes in antibody solution at 35 C antibody solution Stain Cocktail 2 at 35 C 3rd Stain Stain the probe array for 5 minutes in Stain the probe array for 5 minutes in SAPE solution at 35 C SAPE solution Stain Cocktail 1 at 35 C Final Wash 15 cycles of 4 mixes cycle with Wash 15 cycles of 4 mixes cycle with Wash Buffer A at 35 C Holding Buffer N A manual process Fill the probe array with Array Holding Buffer e Wash Buffer A non stringent wash buffer e Wash Buffer B stringent wash buffer chapter 4 Array Washing and Staining 45 Table 4 3 FS450 Fluidics FS400 Fluidics Protocol Protocol 49 FS450_0001 EukGE WS2v5 and add e g Human Tiling 2 0R Array Set Array Holding Buffer 64 FS450_0001 EukGE WS2v5 and add e g Human Promoter 1 0R Array Array Holding Buffer 100 FS450_0002 Midi_euk2v3 and add e g S pombe Tiling 1 0 FR Array Array Holding Buffer W
28. ffymetrix cDNA cleanup columns from the GeneChip Sample Cleanup Module eluting with 20 pL Elution Buffer see Cleanup of Double Stranded DNA on page 57 5 Load 100 500 ng of purified DNA sample on an agarose gel to check sonication efficiency Typically sheared DNA size ranges from 100 4000 bp with the average size fragment between 200 1000 bp chapter 2 Chromatin Immunoprecipitation Assay 19 A B C Jurkat DNA amp Sonication Jurkat DNA amp MNase Ideal DNA Fragmentation amp Sonication Treatment 4000 2000 1000 750 jj 1000 500 400 300 200 100 50 Figure 2 1 A Sheared DNA from HL 60 cells following 8 sonication pulses show the optimal size range for immunoprecipitation 200 1000 bp with the majority of DNA fragments between 300 500 bp Certain cell types may be more resistant to shearing by sonication and would require treatment with Micrococcal nuclease MNase to fragment chromatin B Jurkat cells after 15 pulses of sonication show little fragmentation of crosslinked chromatin C Fragmentation of Jurkat chromatin is achieved with MNase treatment MNase enzyme concentration may have to be titrated based on cell type and density lane1 200U lane2 100U lane3 25U The laddering phenomenon seen with MNase treatment is common due to the specific cleavage of DNA by MNase between nucleosomes NOTE Optional DNA Shearing Method Micrococcal Nuclease Treatment 1 Add ap
29. fied 2 A Terminal Deoxynucleotidyl DNA target Terminal Labeling y Transferase DLR Biotin labeled H gt TITI TTTI sLLILI elll gt Hybridization SAPE Biotinylated Pi anti streptavidin antibody mimar Jil Washing Staining Day 4 Scanning ay Legend DT Bi A Proteins A Antibody Protein A beads LLLLI DNA Figure 1 1 Chromatin Immunoprecipitation Assay Schematic MATERIALS 6 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table 1 1 Materials Required Material Source Part Number Formaldehyde Solution 37 500 mL Sigma Aldrich F8775 Glycine 1 kg Sigma Aldrich 50046 Phosphate Buffered Saline PBS pH 7 4 1X liquid Various IGEPAL CA 630 Sigma Aldrich 9036 19 5 Phenylmethanesulfonyl Fluoride Solution PMSF 250 mL Sigma Aldrich 93482 Microccocal Nuclease MNase Optional USB 70196Y EGTA optional Sigma Aldrich E3889 100G Protease Inhibitor Tablet Roche 11873580001 Decrosslink and check sonication efficiency Proteinase K New England BioLabs P8102S LiCl 8M 500 mL Sigma Aldrich L7026 Glycogen Roche 10901393001 Immunoprecipitation Triton X100 non ionic viscous liquid Roche 10789704001 Protein A Sepharose CL 4B Amersham 17 0963 03 Antibody Various NOTE Antibody should be qualified for chromatin immunoprecipitation See www chiponchip org for a list of qualified antibodies chapter 1 Overview
30. he Fluidics Station dialog box 2 Choose Prime_450 for the respective modules in the Protocol chapter 4 Array Washing and Staining 43 drop down list 3 Change the intake buffer reservoir A to Wash Buffer A non stringent wash buffer and intake buffer reservoir B to Wash Buffer B stringent wash buffer 4 Select the All Modules check box then click Run Procedure C Wash and Stain Probe Arrays After 16 hours of hybridization remove the hybridization cocktail from the probe array and add to any remaining Hybridization Cocktail that was saved in Chapter 3 Procedure A Fill the probe array completely with the appropriate volume of Non Stringent Wash Buffer Wash Buffer A as described in Table 4 1 below Table 4 1 49 250 uL 64 250 uL 100 160 uL If necessary at this point the probe array can be stored at 4 C for up to 3 hours before proceeding with washing and staining Equilibrate the probe array to room temperature before washing and staining This procedure takes approximately 90 minutes to complete 44 Affymetrix Chromatin Immunoprecipitation Assay Protocol FLUIDICS PROTOCOLS Table 4 2 Fluidics Protocols for GeneChip Tiling Arrays Fluidics Station 400 EukGE WS2v5 Fluidics Station 450 FS450_ 0001 Post Hyb Wash 1 10 cycles of 2 mixes cycle with Wash Buffer A at 30 C 10 cycles of 2 mixes cycle with Wash Buffer A at 30 C Post Hyb Wash 2 6 cycles o
31. heared into smaller fragments using sonication Protein DNA complexes are then immunoprecipitated with an antibody directed against the specific protein of interest Following the immunoprecipitation crosslinking is reversed samples are protease treated and the purified DNA sample is amplified using a random primed PCR method Subsequently targets are fragmented and labeled to hybridize onto GeneChip Tiling Arrays By comparing the hybridization signals generated by an immunoprecipitated sample versus an antibody negative or non specific antibody control the regions of chromatin protein interaction can be identified Studies were performed at Affymetrix to evaluate the robustness and sensitivity of the ChIP assay however because of the variability associated with the quality and affinity of various antibodies against their intended targets results may vary from one antibody to the next The procedure outlined in this protocol describes all the necessary steps and reagents for fixing cells fragmenting chromatin immunoprecipitating sheared chromatin amplifying and labeling precipitated DNA We would like to acknowledge Mark Biggin and Xiao Yong Li of the Lawrence Berkeley National Lab for sharing their modifications to the ChIP protocol We have incorporated their improvements to the amplification step page 23 with their approval 4 Affymetrix Chromatin Immunoprecipitation Assay Protocol Chromatin Immunoprecipitation Assay
32. hrough and place the column in a 1 5 mL collection tube 7 Pipet recommended amount of cDNA Elution Buffer directly to the column membrane and incubate at room temperature for 1 minute Then spin at lt 25 000 x g for 1 minute 8 Take 2 pL from each sample to determine the yield by spectrophotometric UV measurement at 260 nm 280 nm and 320 nm Concentration of Double Stranded cDNA pg pL Ago A320 x 0 05 x dilution factor pg DNA eluate in pL x DNA in pg pL 58 Affymetrix Chromatin Immunoprecipitation Assay Protocol Mi SRE BER ae Appendix B Buffers and Solutions Required for ru Hybridization Washing and Staining Appendix B 61 Hybridization Washing and Staining Buffers and Solutions Required for Array Table B 1 Reagents for Hybridization Wash and Satin Material Source P N GeneChip Control Oligo B2 3 nM Affymetrix 900301 Acetylated Bovine Serum Albumin BSA solution 50 mg mL Invitrogen 15561 020 Herring Sperm DNA Promega D1811 R Phycoerythrin Streptavidin Molecular Probes S 866 Goat IgG Reagent Grade Sigma Aldrich 15256 Anti streptavidin antibody goat biotinylated Vector BA 0500 Laboratories Water Molecular Biology Grade Cambrex 51200 5M NaCl RNase free DNase free Ambion 9760G MES hydrate SigmaUltra Sigma Aldrich M5287 MES Sodium Salt Sigma Aldrich M5057 EDTA Disodium Salt 0 5M solution 100 mL Sigma Aldrich E
33. lt Elution Buffer 25 mM Tris HCI made from stock 1M Tris HCI pH 7 5 10 mM EDTA 0 5 SDS chapter 1 Overview 11 MISCELLANEOUS REAGENTS AND SUPPLIES Table 1 3 Miscellaneous Reagents and Supplies Material Supplier Part Number Miscellaneous Reagents Absolute ethanol Gold Shield Chemical Co RNA 6000 Nano LabChip Kit Agilent 5065 4476 Gel Shift Assay Optional Novex XCell SureLock Mini Cell Invitrogen El0001 TBE Gel 4 20 10 mm 12 well Invitrogen EC62252 5X Sucrose Gel Loading Dye Amresco E 274 10X TBE Buffer Cambrex 50843 SYBR Gold Invitrogen S 11494 10 bp DNA ladder and 100 bp DNA ladder Invitrogen 10821 015 and 15628 019 ImmunoPure NeutrAvidin Pierce 31000 PBS pH 7 2 Invitrogen 20012 027 Miscellaneous Supplies 1 2 Agarose Gells Various 1 5 mL RNase free Microfuge Tubes Ambion 12400 1 5 mL Non stick RNase free Microfuge Tubes Ambion 12450 0 2 mL MicroAmp Reaction Tubes 8 tubes strip Applied Biosystems N801 0580 MicroAmp Caps for 8 Strip Tubes Applied Biosystems N801 0535 Pipette for 25 mL VWR 53283 710 Pipet Aid VWR 53498 103 Dolphin nose Tubes Costar Corning 3213 SpinX Columns Costar Corning 8163 MicroSpin S 300 HR Columns GE Healthcare 27 5130 01 12 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table 1 3 Continued Miscellaneous Reagents and Supplies
34. mM dUTP 2 mM Store at 20 C 6 PCR Mix Setup Table 2 4 First round DNA from Step 4 20 pL 10X PCR Buffer 10 uL 25 mM MgCl 3 pL 10 mM dNTPs dUTP 3 75 uL 100 uM Primer Bt 4uL 5 U uL Taq Polymerase 2 uL Nuclease free Water 57 25 uL Total Volume 100 uL Add MgCl if using magnesium free 10X PCR Buffer t Primer B GTTTCCCAGTCACGGTC chapter 2 Chromatin Immunoprecipitation Assay 27 7 Cycle conditions A 15 cycles 1 95 C 30 seconds 2 45 C 30 seconds 3 55 C 30 seconds 4 72 C 1 minute B 15 cycles 1 95 C 30 seconds 2 45 C 30 seconds 3 55 C 30 seconds 4 72 C 1 minute For every subsequent cycle add 5 seconds E g cycle 1 60 seconds cycle 2 65 seconds etc C 4 C hold 8 Check amplified DNA on 1 agarose gel 1 Number of PCR amplification cycles may require optimization OPCR can be used to evaluate enrichment of im munoprecipitated sample 28 Affymetrix Chromatin Immunoprecipitation Assay Protocol 4000 3000 2000 1000 750 500 400 300 200 Figure 2 2 PCR amplified ChIP targets from HL 60 cells immunoprecipitated with an Sp1 antibody Replicate PCR reactions lanes 1 to 3 were performed on the same IP sample and product sizes ranged from 200 bp to over 2 Kb but the actual product sizes may vary depending on original size of sheared chromatin 9 Purify PCR samples with Affymetrix cDNA cleanup columns
35. n 450 gently lift up the cartridge lever to engage or close the washblock The fluidics station automatically performs a Cleanout procedure The LCD window indicates the progress of the Cleanout procedure 3 When the fluidics station LCD window indicates REMOVE VIALS the Cleanout procedure is complete 4 Remove the sample microcentrifuge vial s from the sample holder s 5 If no other arrays are to be processed place wash lines into a bottle filled with deionized water 6 Choose Shutdown or Shutdown_450 for all modules from the drop down Protocol list in the Fluidics Station dialog box Click the Run button for all modules The Shutdown protocol is critical to instrument reliability Refer to the appropriate Fluidics Station User s Guide for more information 7 After Shutdown protocol is complete flip the ON OFE switch of the fluidics station to the OFF position To maintain the cleanliness of the fluidics station and obtain the highest quality image and data possible the bleach protocol is highly recommended Please refer to the GeneChip Fluidics Station 450 250 User s Guide P N 08 0092 available at www affymetrix com Chapter 5 Scanning Chapter 5 WARNING E 51 The GeneChip Scanner 3000 7G is also controlled by GeneChip Operating Software GCOS The probe array is scanned after the wash protocols are complete Make sure the laser is warmed up prior to scanning by turning
36. nstrument dependent It is recommended that conditions are optimized with a single sample prior to scaling up procedures to multiple samples Best sonication conditions at Affymetrix were achieved with a Branson Sonifier 450D using a double step microtip set at 60 duty 50 amplitude 1 minute pulses with 1 minute rest Both pulsing and resting steps were performed in an ice bath 8 to 10 pulses total for HL 60 cells Number of pulses may be dependent on cell density as well as cell type 8 Aliquot the sonicated samples into two 1 5 mL microcentrifuge tubes then microcentrifuge 14 000 rpm 10 minutes at 4 C to remove cellular debris 9 Pool supernatants from Step 8 ina 15 mL conical tube 18 Affymetrix Chromatin Immunoprecipitation Assay Protocol 10 The sonication efficiency can be checked by taking an aliquot 100 pL of this supernatant de crosslinking it see Procedure C below and running the de crosslinked DNA on a 1 2 agarose gel 11 Divide the samples into aliquots equivalent to 5 x 10 cells 1 IP flash freeze and store at 80 C for later use or take straight through the IP Procedure C Check Sonication Efficiency 1 Add 100 pL 10 mM Tris pH 8 0 to the 100 pL aliquot taken from the sonicated samples 2 Add 2 pL Proteinase K 20 mg mL and mix well by vortexing Incubate 42 C for 2 hours then 65 C for 6 hours to overnight This step can be performed in a thermocycler 4 Clean up using A
37. ntrifuge vials into the sample holders 1 2 and 3 on the fluidics station e Place one vial containing 600 pL of SAPE Solution Mix Stain Cocktail 1 in sample holder 1 Place one vial containing 600 pL of Antibody Solution Mix Stain Cocktail 2 in sample holder 2 e Place one vial containing 800 pL Array Holding Buffer in sample holder 3 e Press down on the needle lever to snap needles into position and to start the run The run begins The Fluidics Station dialog boxes at the workstation terminal and the LCD window display the status of the washing and staining as they progress At the end of the run or at the appropriate prompt remove the microcentrifuge vials containing the stain solutions and replace with three empty microcentrifuge vials Remove the probe arrays from the fluidics station modules by first pressing down the cartridge lever to the eject position Check the probe array window for large bubbles or air pockets e If bubbles are present proceed to Table 4 4 Ifthe probe array has no large bubbles it is ready to be scanned on GeneChip Scanner 3000 7G Pull up on the cartridge lever to engage washblock and proceed to Chapter 5 Scanning on page 49 If you do not scan the arrays right away keep the probe arrays at 4 C and in the dark until ready for scanning If there are no more samples to hybridize shut down the fluidics station following the procedure outlined in the section Shut Down the Fluidi
38. pM 2X Hybridization Mixt 100 uL 1X DMSO 14 0 uL 7 Nuclease free Water up to 200 0 uL Total Volume 200 0 uL This volume is 56 HL if a portion of the sample was set aside for gel shift analysis t Available in the GeneChip Hybridization Wash and Stain Kit If preparing buffers see Appendix B for buff er composition 36 Affymetrix Chromatin Immunoprecipitation Assay Protocol Table 3 2 Hybridization Cocktail for use with serial hybridizations e g GeneChip Human Tiling 2 0R Array Set and GeneChip Mouse Tiling 2 0R Array Set Component Volume in 1 Final Concentration Rxn or Amount Fragmented and Labeled DNA Target 60 0 uL 9 0 ug Control Oligonucleotide B2 4 uL 50 pM 2X Hybridization Mix 120 uL 1X DMSO 16 8 uL 7 Nuclease free Water up to 240 0 uL Total Volume 240 0 uL This volume is 58 HL if a portion of the sample was set aside for gel shift analysis Available in the GeneChip Hybridization Wash and Stain Kit If preparing buffers see Appendix B for buff er composition 2 Flick mix and centrifuge the tube Heat the Hybridization Cocktail at 99 C for 5 minutes Cool to 45 C for 5 minutes and centrifuge at maximum speed for 1 minute 4 Inject 200 pL of the specific sample into the array through one of the septa see Figure 3 1 for location of the septa on the array Save the remaining hybridization cocktail in 20 C for future use 5 Place arra
39. propriate units of MNase based on prior optimization of MNase to effectively shear crosslinked chromatin This can range from 25 U to 200 U or more for each IP performed 2 Incubate at 37 C 10 minutes 3 Add 30 pL 200 mM EGTA to stop the reaction 20 Affymetrix Chromatin Immunoprecipitation Assay Protocol Procedure D Incubate With Specific Antibody 1 If the sample from Procedure B Step 11 on page 18 was frozen thaw Transfer supernatant to a 15 mL tube and add 5 volumes of IP dilution buffer containing protease inhibitors tablet from Roche add before use Pre equilibriate protein A Sepharose beads by washing 100 pL beads with 1 mL IP dilution buffer pellet cells by centrifuging for 2 minutes at 2 000 rpm at 4 C in a microcentrifuge Remove 800 pL supernatant Pre clear chromatin by adding 200 pL pre equilibrated Protein A Sepharose beads Incubate on a rotating platform at 4 C for 30 minutes Centrifuge at 2 000 rpm for 2 minutes at 4 C in a swinging bucket rotor Transfer supernatant to a new 15 mL tube and discard beads Add 10 to 15 pg of antibody per IP Usually a negative control is performed using the same number of cells with a non specific IgG or no antibody mock IP control The amount of antibody to be added is dependent on quality affinity specificity and type of antibody used Users may have to titrate the amount of antibody used for each IP 9 Incubate on rot
40. resses 54 Affymetrix Chromatin Immunoprecipitation Assay Protocol A Eni ai PE no m lai a Appendix Cleanup of Double Stranded DNA Appendix A 57 rl Cleanup of Double Stranded DNA This Procedure requires the use of the GeneChip Sample Cleanup Module 1 If not already done add 24 mL of Ethanol 100 to the DNA Wash Buffer supplied in the GeneChip Sample Cleanup Module 2 Add 5X volumes of cDNA Binding Buffer to sample and vortex for 3 seconds The GeneChip Sample Cleanup Module includes 20 mL of cDNA Binding Buffer In order to process 30 samples following the Affymetrix Chromatin Immunoprecipitation Assay Protocol additional cDNA Binding Buffer is required This buffer should be purchased directly from QIAGEN When purchasing the cDNA Binding Buffer from OIAGEN please order ERC Buffer part number 1018144 3 Apply the sample to a cDNA Spin Column sitting in a 2 mL Collection Tube max capacity of column 700 pL if volume exceeds 700 pL spin 700 pL at gt 8 000 x g for 1 minute discard flow through and repeat Spin at gt 8 000 x g for 1 minute Discard the flow through 5 Transfer the cDNA Spin Column to a new 2 mL Collection Tube and add 750 pL of cDNA Wash Buffer to the column Spin at gt 8 000 x g for 1 minute and discard the flow through 6 Open cap of the cDNA Spin Column and spin at lt 25 000 x g for 5 minutes with the caps open Discard the flow t
41. rl 200 mL culture or invert tube containing 20 mL of adherent cells occasionally to mix cells Add 1 20 volume 2 5 M glycine and incubate at RT 5 minutes with gentle mixing to quench formaldehyde reaction Perform remaining steps on ice Pellet cells at 4 C 300 500g 4 minutes and discard supernatant in formaldehyde waste Wash pellet with 10 mL ice cold 1X PBS to resuspend cells and transfer to 15 mL tube Pellet cells at 4 C 300 500g 4 minutes and discard supernatant and repeat wash with ice cold 1X PBS once Wash the pellet 3 times with 10 mL Lysis Buffer with fresh PMSF Pellet cells at 300 500g 5 minutes between washes Discard supernatant and proceed to the next step or flash freeze pellet and store at 80 C Resuspend the pellet in 1 mL pre IP dilution buffer add 60 pL PMSF and bring final reaction volume to 1 5 mL with pre IP dilution buffer chapter 2 Chromatin Immunoprecipitation Assay 17 6 Add to the tube Table 2 1 100 mM PMSF 40 uL 25X Protease Inhibitor Stock 100 uL Pre IP Dilution Buffer 460 uL 20 SDS 100 uL 5 M NaCl 80 uL Nuclease free Water 220 uL Final Sample Volume Before Sonication 2 5 mL if using optional MNase see details on page 19 7 Sonicate sample to lyse cells and shear DNA to 100 1000 bp fragments Some cell types e g Jurkat may require optional MNase treatment See page 19 for details Optimized shearing conditions are cell type and i
42. y in 45 C hybridization oven at 60 rpm and incubate for 16 hours 6 After hybridization remove the hybridization cocktail for future use chapter 3 Hybridization and Array Processing 37 Plastic cartridge Front Probe array on glass substrate GeneChip Probe Array Figure 3 1 GeneChip Probe Array 38 Affymetrix Chromatin Immunoprecipitation Assay Protocol Chapter 4 Array Washing and Staining Chapter 4 41 Procedure A Enter Experiment Information To wash stain and scan a probe array an experiment must first be registered in GeneChip Operating Software GCOS Please follow the instructions detailed in the Setting Up an Experiment section of the GCOS User s Guide The fields of information required are Experiment Name Probe Array Type For multi array sets please refer to the design number indicated on the array cartridge label Sample Name Sample Type Project Sample templates experiment templates and array barcodes can also be employed in GCOS to standardize and simplify the registration process Please see the Affymetrix GeneChip Operating Software User s Guide PIN 701439 for more information The Project Sample Name and Experiment Name fields establish a sample hierarchy that organizes GeneChip data in GCOS In terms of the organizational structure the Project is at the top of the hierarchy followed by Sample Name
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