User Guide
Contents
1. sheared DNA size ranges from 100 4000 bp with the average size fragment between 200 1000 bp 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 A Ideal DNA Fragmentation Ladder 4000 2000 1000 750 j 500 400 300 200 100 50 B Jurkat DNA amp Sonication C Jurkat DNA amp Sonication amp MNase Treatment NOTE Optional DNA Shearing Method Micrococcal Nuclease Treatment 1 Add appropriate units of MNase based on prior optimization of MNase to effectively shear crosslinked chromatin This can range from 25U to 200U or more for each IP performed 2 Incubate at 37 C 10 minutes 3 Add 30 pL 200 mM EGTA to stop the reaction Procedure D Incubate With Specific An2. 0R Array Setup 3 reactions for multi array sets e g Human Tiling 2 0R Array Set Table 2 2 Component Volume for 1 Rxn Purified DNA 10 uL 5X Sequenase Reaction Buffer 4 uL Primer A 200 uM 4 uL Total Volume 18 uL Included with enzyme tPrimer A GTTTCCCAGTCACGGTC N HPLC purified 3 Cycle conditions Random priming A 95 C for 4 minutes Snap cool samples on ice 10 C hold Prepare first cocktail Table 2 3 onm Chapter 2 Chromatin Immunoprecipitation Assay 16 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 uL per sample F Mix well by pipetting and put the sample back in thermocycler block G 10 C for 5 minutes H Ramp from 10 C to 37 C over 9 minutes O IYO 2ZEB FTA TET 2 37 C for 8 minutes 95 C for 4 minutes Snap cool on ice 10 C hold Add 1 0 uL of 1 3U uL Sequenase 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 4 For each IP purify with Microspin S 300 HR GE Healthcare columns 2 columns per reaction as follows A B C E F G Add 20 uL of 10 mM TE pH 8 0 to each reaction Spin 2 columns A amp B at 3 000 rpm for 1 minute discard f
3. Buffer and 200 uL beads for each IP d sample Centrifuge 2 000 rpm 2 minutes at 4 C Discard around 800 uL supernatant save 400 uL of beads in buffer at the bottom of the tube Transfer 400 uL beads to each sample Add PMSF to each tube sample final concentration 1mM PMSF 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 uL 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 uL 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 uL ChIP wash 3 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 uL 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 uL Elution Buffer to the column Incubate at 65 C for 30 minutes Centrifuge at 3 000 rpm at RT for 2 minutes Add 200 uL Elution Buffer to the column Centrifuge at 3 000 rpm
4. DNA Target 60 0 uL 7 5 ug Control Oligonucleotide B2 3 3 uL 50 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 uL if a portion of the sample was set aside for gel shift analysis t Available in the GeneChip Hybridization Wash and Stain Kit 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 Mixt 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 uL if a portion of the sample was set aside for gel shift analysis t Available in the GeneChip Hybridization Wash and Stain Kit Flick mix and centrifuge the tube 3 Heatthe 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 uL 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 array in 45 C hybridization oven at 60 rpm and incubate for 16 hours 6 After hybridization remove the hybridization cocktail
5. at RT for 2 minutes This 400 uL eluted sample is the enriched or IP d sample Chapter 2 Chromatin Immunoprecipitation Assay 15 Procedure F Reverse Crosslinks 1 Add 5 uL Proteinase K 20mg mL per 100 uL of negative control or IP sample mix well 20 uL for 400 uL of eluted sample 2 Incubate in incubator at 65 C overnight DAY 3 Procedure G Cleanup De crosslinked Samples 1 USB PrepEase DNA Clean Up Kit see Cleanup of Double Stranded DNA on page 23 NOTE 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 obtained 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 L working stock will be needed for each sample being amplified 1 Use 10 uL of IP d or negative control sample for initial round of linear amplification 2 Setup first round reaction Set up reaction for single array products e g Human Promoter 1
6. for future use Chapter 3 Hybridization and Array Processing 22 Figure 3 1 GeneChip Probe Array Plastic cartridge Front Probe array on glass substrate Procedure B Array Wash Stain and Scan For instructions on array washing staining and scanning please refer to the GeneChip Expression Wash Stain and Scan User Manual P N 702731 Cleanup of Double Stranded DNA Cleanup of Double Stranded DNA This Step requires the use of the PrepEase DNA Clean Up Kit PrepEase DNA Clean Up Kit P Ns 78758 78759 Brief Protocol for Concentration Desalination and or Removal of Enzymes Lu IMPORTANT Check that ethanol was added to NT3 Buffer before starting 1 Adjust DNA binding conditions A Add 5 volumes of N2P Buffer to 1 volume of sample e g 500 uL N2P Buffer and 100 uL sample B Mix well Bind DNA sample to column A Place PrepEase Clean Up Column into a 2 mL PrepEase Collecting Tube B Pipet the sample directly into the center of the column C Centrifuge 1 min at 11 000 x g D Discard flow through Wash column A Add 600 uL NT3 Buffer to column B Centrifuge 1 min at 11 000 x g C Discard flow through Place column back into collecting tube Dry column Centrifuge 2 min at 11 000 x g Elute DNA A Place the column into a clean 1 5 ml microcentrifuge tube Add 15 50 uL NE Buffer to column Incubate at room temperature for 1 min Un D Centrifuge 1 mi
7. of 2 x 10 cells Procedure B Fix Cells Lyse and Sonicate Whole Cell Extracts DAY 1 NOTE Centrifugation steps involving cells are best performed with a swing bucket type rotor Adherent Cells NOTE End users may optimize the sequence of fixing and harvesting cells to minimize the degree to which cell physiology is disrupted 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 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 Suspension Cells 1 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 swirl 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 i
8. DNA Clean Up Kit USB 78758 Hybridization Stain and Wash GeneChip Hybridization Wash and Stain Kit Affymetrix 900720 Control Oligonucleotide B2 33M Affymetrix 900301 Buffers Table 1 2 Buffers Lysis Buffer Store at 4 C 10 mM Tris HCl made from stock 1M Tris HCl pH 7 5 10 mM Nacl 3 mM MgCl 0 5 IGEPAL 1 mM PMSSF add fresh Pre IP Dilution Buffer Store at RT 10 mM Tris HCl made from stock 1M Tris HCl 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 HCl made from stock 1M Tris HCl pH 8 2 mM EDTA 196 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 HCl made from stock 1M Tris HCl pH 8 2 mM EDTA 196 Triton X 100 150 mM NaCl 1 mM PMSF add fresh ChIP Wash 2 Store at RT 20 mM Tris HCl made from stock 1M Tris HCl pH 8 2 mM EDTA 196 Triton X 100 0 196 SDS 500 mM NaCl 1 mM PMSSF add fresh ChIP Wash 3 Store at RT 10 mM Tris HCl made from stock 1M Tris HCl pH 8 1 mM EDTA 0 25M LiCl 0 5 IGEPAL 0 5 Deoxycholate sodium salt Elution Buffer 25 mM Tris HCI made from stock 1M Tris HCI pH 7 5 10 mM EDTA 0 5 SDS Chapter 1 Overvi
9. 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 PCR Amplification Sequenase Version 2 0 DNA Polymerase USB 70775Y Primer A 200 uM 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 Table 1 1 Materials Required Continued Chapter 1 Overview 7 Material Source Part Number Wash Buffer Tris HCl Various EDTA Various SDS 100g Sigma Aldrich 71725 NaCl Various Deoxycholate sodium salt 100g Sigma Aldrich D6750 MgCl 1M Various CaCl 1M Sigma Aldrich 21115 Fragmentation and Labeling Uracil DNA Glycosylase UDG 2 U uL USB 71960 Human Apurinic Apyrimidinic Endonuclease 1 APE 1 USB 78454 Includes 10X APE 1 Reaction Buffer Terminal Deoxynucleotidyl Transferase rTdT Recombinant 30 U uL USB 72033 5X TdT buffer included DNA Labeling Reagent DLR 10 mM USB 79015 DNA Cleanup PrepEase
10. TRG Affymetrix ilser Guide Affymetrix Chromatin Immunoprecipitation Assay Protocol For research use only Not for use in diagnostic procedures Trademarks Affymetrix Axiom Command Console CytoScan DMET GeneAtlas GeneChip GeneChip compatible GeneTitan Genotyping Console myDesign NetAffx OncoScan Powered by Affymetrix PrimeView Procarta and QuantiGene are trademarks or registered trademarks of Affymetrix Inc USB the logo design and PrepEase are registered trademarks of USB Corporation 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 p
11. ase treat Day 2 IP d DNA CI Sample Cleanup K IP d DNA Primer Adapter Sequenase dNTPs Linear Random Adapter I Amplification I 511101 sH Adapter Primer dNTP dUTP Taq polymerase I PCR amplify and incorporate 4 dUTP U Uracil DNA Fragmentation 2 of APE 1 Til 11111 np L LIII Glycosylase L Day 3 Fragment and label amplified r Terminal Deoxynucleotidyl DNA target Terminal Labeling A Transferase DLR Biotin labeled e iit TTTTI NENNEN oLLLI Hybridization SAPE Biotinylated lt anti streptavidin antibody Hybridize arrays gt Washing Staining v Day4 Scanning 2y Legend TIO E A Proteins A Antibody Protein A beads LLLLL DNA Materials Table 1 1 Materials Required Chapter 1 Overview 6 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
12. atents U S Patent Nos 5 445 934 5 744 305 5 945 334 6 140 044 6 399 365 6 551 817 6 733 977 7 629 164 7 790 389 and D430 024 and other U S or foreign patents Products are manufactured and sold under license from OGT under 5 700 637 PrepEase products are covered under European Patent EP 0496822 and US Patent 6 428 703 Copyright Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Appendix A Appendix B Overview S ars nes ge base KR uo kk kk kk kk kk kk kk wt kk kk kk kk kk kk kk kk kk kk kk 4 llrstelllal JN gDww y 2n2a2x2x2nm pPnmnnnnDDDb_____n_mrm nnbs ae m 4 Chromatin Immunoprecipitation Assay Protocol Optimization WW RR KK KK SJ 4 FC i rg ETE TTE eee 6 BONS Set fcc oe ait oe ue etn eee Sed yy ad Sia ease oie Ont Bed er 8 Miscellaneous Reagents and Supplies kk A kk kk kk KK KK KK KK KK KK KK KK KK KK KI KK 9 Chromatin Immunoprecipitation Assay 000 KK eee KK RR KK 11 Procedure Az Prepare Cells s A haa dt bbe nb Sok Wn anin d bak Dek dodged here 4 11 Procedure B Fix Cells Lyse and Sonicate Whole Cell Extracts AK RR 11 Adherent Cells vs assent eee hi tee eee HHHH He 11 Suspension Cells 2s sede xema eset he eed bea e s e vepres ied s 11 WashuCell Pellet 4 2 iret geriami Teti beh IE UD AE ek eed dard 12 Procedure C Check Sonication Efficiency kk kk kk kk kK KK KK KK KK ees 12 Procedure D Incubate With Specific Antibody kk kk k
13. ce Pellet cells at 4 C 300 500g 4 minutes and discard supernatant in formaldehyde waste Chapter 2 Chromatin Immunoprecipitation Assay 12 Wash Cell Pellet 1 Wash pellet with 10 mL ice cold 1X PBS to resuspend cells and transfer to 15 mL tube 2 Pellet cells at 4 C 300 500g 4 minutes and discard supernatant and repeat wash with ice cold 1X PBS once 3 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 5 Resuspend the pellet in 1 mL pre IP dilution buffer add 60 uL PMSF and bring final reaction volume to 1 5 mL with pre IP dilution buffer 6 Add to the tube Table 2 1 Component Volume for 1 Rxn 100 mM PMSF 40 uL 25X Protease Inhibitor Stock 100 uL Pre IP Dilution Buffer 460 uL 2096 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 13 7 8 9 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 13 for details NOTE Optimized shearing conditions are cell type and instrument dependent It is recommended that conditions are optimized with a single sample prior to scaling up procedures to multiple samples Best sonication condi
14. 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 sheared 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
15. ew Miscellaneous Reagents and Supplies Table 1 3 Miscellaneous Reagents and Supplies Chapter 1 Overview 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 E10001 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 1 296 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 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 Hybridizati
16. k kK KK KK KK KEK KK KK KK 13 Procedure E Immunoprecipitate and Wash Wak kk kk kK eee 14 Procedure Fs Reverse Cr sSlinKS xeu sasa erdina ea dee ea sabes SER eed 15 Procedure G Cleanup De crosslinked Samples n on nananana kk 00 eee 15 Procedure H PCR Amplify Immunoprecipitated DNA Targets WR KK RR KIR 15 Procedure I Fragment Amplified Targets kk kk kk kk kK KK KK KK KK KK KK K KI KIR K IK IK 19 Procedure J Label Fragmented Double Stranded DNA AW KK ee 20 Hybridization and Array Processing n nenna aannaaien 21 Procedure A Hybridize Labeled Target on the Arrays WWW KK KK KK K R RR KIRR KI YI 21 Procedure B Array Wash Stain and Scan kk kk kk kk KK KI KK KIRI K KI RIK KI K KIRI KIR KI KIRI K KK 22 Cleanup of Double Stranded DNA kk kk kk kK KK KK KK eee 23 Cleanup of Double Stranded DNA Aka kk kk kK KK KI KK KK KK KK KIRI e 23 Brief Protocol for Concentration Desalination and or Removal of Enzymes 23 Brief Protocol for PCR Purification lt j llle 23 Brief Protocol for DNA Purification from Chromatin Immunoprecipitation ChIP Assay 24 Contact Information ou 64 oce Qe ow are oc RCM k aha xal kla Oe kula RR CD CR CCS CES 25 Overview 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
17. low through Transfer reaction volume 43 uL to column A while equilibrating column B with 300 uL of 10 mM Tris pH 8 0 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 Transfer flow through of column A to column B with new collection tube Spin at 3 000 rpm for 2 minutes Collect 56 uL of first round purified DNA per reaction 5 Prepare dNTP dUTP mix Chapter 2 Chromatin Immunoprecipitation Assay 17 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 mM dUTP 2 mM Store at 20 C 6 PCR Mix Setup Table 2 4 Component Volume for 1 Rxn First round DNA from Step 4 20 uL 10X PCR Buffer 10 uL 25 mM MgCl2 3 uL 10 mM dNTPs dUTP 3 75 uL 100 uM Primer Bt 4 uL 5 U uL Taq Polymerase 2yL Nuclease free Water 57 25 uL Total Volume 100 uL Add MgCl if using magnesium free 10X PCR Buffer Primer B GTTTCCCAGTCACGGTC 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
18. min at 11 000 x g 6 Take 2 uL from each sample to determine the yield by spectrophotometric UV measurement at 260 nm 280 nm and 320 nm Concentration of Double Stranded cDNA ug uL A A320 x 0 05 x dilution factor ug DNA eluate in uL x DNA in ug uL Contact Information Affymetrix Inc 3420 Central Expressway Santa Clara CA 95051 USA Email 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 Email supporteurope affymetrix com UK and Others Tel 44 0 1628 552550 France Tel 0800919505 Germany Tel 01803001334 Fax 44 0 1628 552585 Affymetrix Japan K K ORIX Hamamatsucho Bldg 7F 1 24 8 Hamamatsucho Minato ku Tokyo 105 0013 Japan Email supportjapan affymetrix com Tel 81 3 6430 4020 Fax 81 3 6430 4021 USB Corporation 26111 Miles Road Cleveland Ohio 44128 USA www usbweb com
19. n Assay 19 Procedure I 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 Fragmentation of ds cDNA Component Volume Amount in 1 Rxn ds cDNA 7 5 ug Nuclease free Water up to 32 2 uL 10X APE 1 Reaction Buffer 4 8 uL Uracil DNA Glycosylase UDG 2U pL 4 0 uL Human Apurinic Apyrimidinic Endonuclease 1 7 0 uL APE 1 10 U uL Total Volume 48 0 uL Table 2 6 Fragmentation Mix for multi array sets e g Human Tiling 2 0R Array Set Fragmentation of ds cDNA Component Volume Amount in 1 Rxn ds cDNA 9 0 ug Nuclease free Water up to 32 2 uL 10X APE 1 Reaction Buffer 4 8 uL Uracil DNA Glycosylase UDG 2U pL 4 0 uL Human Apurinic Apyrimidinic Endonuclease 1 7 0 uL APE 1 10 U uL Total Volume 48 0 uL 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 u 37 C for 1 hour 93 C for 2 minutes u 4 C for at least 2 minutes Flick mix spin down the tubes and transfer 45 uL 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 no
20. n at 11 000 x g Brief Protocol for PCR Purification Lu IMPORTANT Check that ethanol was added to NT3 Buffer before starting 1 Adjust DNA binding conditions A Add 5 volumes of N2P Buffer to 1 volume of sample e g 250 uL N2P Buffer and 50 uL sample B Mix well Continue with Step 2 to Step 5 of the Brief Protocol for Concentration Desalination and or Removal of Enzymes on page 23 Appendix A Cleanup of Double Stranded DNA 24 Brief Protocol for DNA Purification from Chromatin Immunoprecipitation ChIP Assay Lu IMPORTANT Check that ethanol was added to NT3 Buffer before starting 1 Adjust DNA binding conditions A Add 5 volumes of N2P Buffer to 1 volume of sample e g 1000 uL N2P Buffer and 200 uL sample Mix well 2 Bind DNA sample to column mon gt Place PrepEase Clean Up Column into a 2 ml PrepEase Collecting Tube Pipet 700 uL of the sample directly into the center of the column Centrifuge 1 min at 11 000 x g Discard flow through Repeat Step B to Step D for the remaining sample 3 Wash column A B C Add 600 uL NT3 Buffer to column Centrifuge 1 min at 11 000 x g Discard flow through Place column back into collecting tube 4 Dry column Centrifuge 2 min at 11 000 x g 5 Elute DNA A B C D Place the column into a clean 1 5 ml microcentrifuge tube Add 30 40 uL NE Buffer to column Incubate at room temperature for 1 min Centrifuge 1
21. ntibody affinities and avidities can vary so the amount of antibody may need to be titrated to achieve optimal sample enrichment 4 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 Chapter 1 Overview 5 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 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 Figure 1 1 Chromatin Immunoprecipitation Assay Schematic Fix cells to HE E crosslinkDNA A a to protein TI r Sonicate to lyse E M EH a cells and shear 4 Lj II P lt I chromatin Day 1 Take small aliquot to decrosslink and check e sonication efficiency Immunoprecipitate E E main sample with 4 selected antibody s NN y Couple to Protein A beads and wash to 7 purify IP d DNA Decrosslink and protein
22. on 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 9 Table 1 3 Miscellaneous Reagents and Supplies Continued Chapter 1 Overview Material Supplier Part Number 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 pL Rainin L 200 Pipette for 100 to 1 000 uL Rainin L 1000 Or equivalent instrument supplies 10 Chromatin Immunoprecipitation Assay Procedure A Prepare Cells 1 Grow enough cells for the number of immunoprecipitation IP reactions to be performed usually 5 x 10 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 group in the downstream two sample analysis Use 0 5 2 x 108 cells per IP For example grow 200 mL of 1 x 10 cells mL for a total
23. 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 on page 15 with their approval Chromatin Immunoprecipitation Assay 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 Experimental Biology cell types proteins of interest antibodies a 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 2 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 3 Antibody Titration A
24. 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 QPCR can be used to evaluate enrichment of immunoprecipitated sample Chapter 2 Chromatin Immunoprecipitation Assay 18 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 123 4000 3000 2000 1000 750 500 400 300 200 9 Purify PCR samples using USB PrepEase DNA Clean Up kit see Cleanup of Double Stranded DNA on page 23 10 Measure DNA using a NanoDrop or other UV vis spectrophotometer Normally greater than 9 ug of amplified DNA is obtained from each reaction NOTE 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 Immunoprecipitatio
25. t labeling the samples immediately store the fragmented DNA at 20 C Chapter 2 Chromatin Immunoprecipitation Assay 20 Fluorescence Migration Time 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 Stranded DNA Labeling Mix 5x TdT Reaction Buffer 12 uL Terminal Deoxynucleotidyl Transferase rTdT 2 uL Recombinant 30 U uL DNA Labeling Reagent DLR 10 mM 1puL Total Volume 15 uL 2 Add 15 uL ofthe Double Stranded DNA Labeling Mix to the DNA samples flick mix and spin them down 3 Incubate the reactions at 37 C for 60 minutes 70 C for 10 minutes 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 Hybridization and Array Processing Procedure A Hybridize Labeled Target on the Arrays This Procedure requires the use of the GeneChip Hybridization Wash and Stain Kit P N 900720 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
26. tibody 1 Ifthe sample from Procedure B Step 11 on page 12 was frozen thaw 2 Transfer supernatant to a 15 mL tube and add 5 volumes of IP dilution buffer containing protease inhibitors tablet from Roche add before use 3 Pre equilibriate protein A Sepharose beads by washing 100 uL 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 uL supernatant 4 Pre clear chromatin by adding 200 uL pre equilibrated Protein A Sepharose beads po CAN ERE an Chapter 2 Chromatin Immunoprecipitation Assay 14 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 ug 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 NOTE 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 Incubate on rotating platform at 4 C overnight or for at least 3 hours at RT DAY 2 Procedure E Immunoprecipitate and Wash 1 SY OF 9 WM 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Pre equilibrate protein A Sepharose beads by adding 1 mL IP Dilution
27. tions 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 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 Pool supernatants from Step 8 in a 15 mL conical tube 10 The sonication efficiency can be checked by taking an aliquot 100 uL 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 107 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 uL 10 mM Tris pH 8 0 to the 100 uL aliquot taken from the sonicated samples 2 Add 2 uL 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 USB PrepEase DNA Clean Up Kit see Cleanup of Double Stranded DNA on page 23 Chapter 2 Chromatin Immunoprecipitation Assay 13 5 Load 100 500 ng of purified DNA sample on an agarose gel to check sonication efficiency Typically
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