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1. Computer is not receiving power Check that the power cord is connected to Computer cannot be switched on the computer and to the wall power outlet SX 8G IP Star shows no movement SX 8G IP Star is not switched on Check that the SX 8G IP Star is switched when a protocol is started on The pipettor head may have lost its home position In the Software select Manual Operation Home After confirming that the pipettor head moves to the home position run the protocol again SX 86 IP Star shows abnormal movement when a protocol is started Dripping Is acceptable when ethanol is being handled For other liquids air is leaking from the syringe pumps Grease or replace the O rings If the problem persists contact DIAGENODE Technical Services Aspirated liquid drips from the disposable tips Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 34 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Technical Assistance amp Ordering Information At DIAGENODE we pride ourselves on the quality and availability of our technical support Our Technical Services Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of DIAGENODE products If you have any questions or experience any difficulties regarding the SX 8G IP Star or DIAGENODE products In general do not hesitate to contact us DIAGE2. screen Yes Mm No Returns the user to the display of the Configuration E screen e he user presses the Text box The screen will be changed to Keyboard or Speed list menu Speed list menu Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 16 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Layout information Tip Rack DN70 Sample number 8 MIMyel be Reagent information o A DIB Elution buffer Screen Layout Information After the user presses the next button from Sample number screen or Configuration screen the Layout Information screen appears Buttons The user presses the Back button The user returns to the previous screen The user presses the Next button The screen shall be changed to Set confirmation When the user presses a block that block is magnified on the work surface layout background The magnified view provides a better display of the correct method setup for that block on the work surface Based on the selected protocol the user follows the indications provided in the screens to set up correctly the different reagents and samples Screen Layout Information Beads Wash Buffer ChIP Buffer H W BID Elution buffer DIB or Elution Buffer B Beads wash buffer A IP wash 1 Wash Buffer H1 C F IP wash No 1 4 g IP Wash 2 Wash Buffer H2 Block Regent Tip Rack DI
3. 10 ul of TE That corresponds to the purified DNA from the sheared chromatin Several DNA pellets can be pooled at this step to have DNA corresponding to a minimum of 60 000 cells in 10 ul of TE 16 Run samples 10 ul of DNA 2 ul of 6x loading dye in a 1 5 agarose gel along with DNA size marker to visualise shearing efficiency 1 Figure 3 Hela cells were fixed with 1 formaldehyde for 10 minutes at RT Cell lysis are performed using the Lysis Buffer tL1 of the Diagenode True MicroChIP kit Samples corresponding to 10 000 cells are sheared during 5 rounds of 5 cycles of 30 seconds ON 30 seconds OFF with the Bioruptor Plus combined with the Bioruptor Water cooler Cat No BioAcc cool at HIGH power setting position H For optimal results samples are vortexed before and after performing 5 sonication cycles followed by a short centrifugation at 4 C 10 ul of DNA equivalent to 60 000 cells are analysed on a 1 5 agarose gel lane 1 lane M 100 bp DNA Molecular Weight Marker ma el Sere e ae Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 33 Troubleshooting Guide Error Cause Remedy SX 8G IP Star is not receiving power Check that the power cord is SX 86 IP Star cannot be switched on connected to the workstation and to the wall power outlet
4. 2 Input If you set eltetetctateiel Screen Running status This screen gives informations about the current running step of the protocol The user can check through this screen the passed and remaining time of the experiment diagenotie B ChIP IPure CAUTION e Add input sample in well 1 and 4 ul 5M NaCl in wells 1 and 12 e After that put caps on tubes diagengte Screen Elution INPUT is defined as a 100ul protocol INPUT 1 pul diluted sheared chromatin 99 pl Elution Buffer b 200 ul protocol INPUT 2ul diluted sheared chromatin 94 ul Elution Buffer Screen Finish End When the protocol is complete a window appears telling user the run is over The screen behind this window should be the Startup screen When OK is pressed then the Startup screen appears and the user can immediately begin to remove their sample and prepare the next run At this point user Is expected to be ready to press RUN Buttons e The user presses the OK button Then screen shall be changed to Categories Name Protocol List Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 19 CAUTION Screen Caution When the protocol finishes the user can return to the protocol list screen A or warm the Do you want to start again a p
5. and F Elution module as follow Reagents Volume Buffer D 96 ul Buffer E 10 ul Buffer F 4 ul Total volume 110 ul Add 100 ul of the Complete Elution Buffer to each chromatin sample Mix thoroughly and incubate samples at 65 C for 4 hours or overnight Extract DNA once with an equal volume of phenol chloroform isoamyl alcohol 25 24 1 Incubate the samples at RT for 10 minutes on a rotating wheel Centrifuge for 2 minutes at 14 000 xg 13 000 rpm at RT Transfer the top aqueous phase into a new 1 5 ml tube 10 Add 1 volume of chloroform isoamyl alcohol 24 1 Incubate the samples at RT for 10 minutes on a rotating wheel 11 Centrifuge for 2 minutes at 14 000 x g 13 000 rpm at RT Transfer the top aqueous phase into a new 1 5 ml tube 12 Precipitate the DNA by adding 40 ul of meDNA precipitant 5 ul of meDNA co precipitant and 1 ml 100 cold ethanol to the sample Incubate at 80 C for 30 minutes Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 32 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL 13 Centrifuge for 25 minutes at 14 000 x g 13 000 rpm at 4 C Carefully remove the supernatant and add 500 ul of ice cold 70 ethanol to the pellet 14 Centrifuge for 10 minutes at 14 000 x g 13 000 rpm at 4 C Carefully remove the supernatant leave tubes opened for 30 minutes at RT to evaporate the remaining ethanol 15 Resuspend the pellet in
6. directory location of the ChIP protocols 3 Start now SX 8G V532 software through SX 8G V52 exe file 4 Press button for Easy Protocol Start screen and load the protocol of interest Before starting the protocol a start confirmation window will appear Press OK and the protocol will run Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 26 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Alternatively incubation time for antibody coating and temperature and incubation time for the IP reaction can be adjusted in an existing protocol by selecting the modify button The modified protocol can also be saved as new protocol SX8G V5 Ver0 7 CHIP_Small_Demo HLD he program ended successfully dlagendiie Modify Parameter for Chir If running ChIP 16 protocol setup half of the incubation time It will incubate half of the time on each block but total time will be correct For instance if you want 10h incubation you have to setup 5h Save modification protocol Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL 7 The program will run through the following steps magnetic bead washes IP and IP washes 14 12 14 16 Pitt tty Antibody immobilize to beads gt IP reaction DNA purificatia
7. in the automated systems are 100 ul and 200 ul Note 2 In general Diagenode does not recommend to preblocked Diagenode magnetic beads BSA is however provided in this kit for customers that would like to proceed with preblocking or to add BSA In the immunoprecipitation reaction For preblocking proceed as follow 1 Pippette 100 ul of beads Add 390 ul CHIP Buffer H and 10 ul BSA 5 0 1 final concentration 2 Incubate 60 minutes by rotation 3 Washed once with 100 ul of ChIP BUffer H 4 Resuspend your beads in 100 ul ChIP Buffer H 10 ul of magnetic beads will bind up to 2 5 ug of Antibody Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 22 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Dispense prepared reagents into the corresponding tubes see picture below ChIP Direct Method Ab coating With this method the antibody is first coated on the surface of the magnetic beads and after that the bound antibodies are added to the sheared chromatin 2 Preparation Buffer Ab Antibody x pl ChIP Buffer H 100 x Antibody coating Elution buffer Input or DIB ChIP reaction Bead washes Elution buffer IP or DIB 1 2 3 4 5 16 7 8 9 10 11 12 gt dD dD dD lt D gt a y lt gt DD lt a A A IPURE DIB Mae ae sae eee onal ee en 1 Elution buffer H 5M NaCl 95 ul 4ul 94 ul 4 ul D
8. of antibody per IP The binding capacity of 10 ul of magnetic beads is 3 ug of antibody If you plan to use more than 3 pg of antibody per IP we recommend that the quantity of beads Is adjusted accordingly Please contact us for advice If required NOTE 1 If required add 2 ul of BSA 5 to the sheared chromatin mix when running ChIP 100 ul protocols or 4 ul of BSA 5 when running ChIP 200 ul protocols NOTE 2 If required NaBu HDAC inhibitior 20mM final concentration or other inhibitors can also be added to the sheared chromatin mix PAGE 23 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 24 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Running protocol Be sure that the computer connected to the SX 8G IP Star never switches to the standby modus standby modus has to be inactivated Standby of the computer will lead to the abort of the protocol Table 3 Protocol Name ChIP DIB 8 protocol ChIP IPure 8 protocol Reagent Preparation Th 1h Magnetic Bead Washes 30 min 30 min Ab coating Ab coating time Ab coating time Immunoselection IP reaction time IP reaction time Washes and elution 30 min Th Add reagents 15 min 15 min DNA isolation or reverse cross linking 30 min DNA isolation 4h reverse cross linking DNA recovery ss DNA ds DNA Input required is sheared chromatin ready to ChIP Note Hands on work time is reduced to 1h45 m
9. 20010 1 ml 20 C Protein A coated paramagnetic beads C03010020 220 The beads are supplied in solution 220 ul 4 C C03010020 660 with detergent and 0 02 sodium 660 ul Do not freeze C03010020 150 azide 1500 ul Protein G coated paramagnetic beads C03010021 220 The beads are supplied in solution 220 ul A C03010021 660 with detergent and 0 02 sodium 660 ul Do not freeze C03010021 150 azide 1500 pl Rabbit IgG C15410206 1 ug pl 250 pl 4 C Mouse IgG C15200001 1 ug ul 15 pl 4 C Antibodies 5 www diagenode com Primer pairs 5uM each Rv amp Fw www diagenode com Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 10 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Table 3 Kits and Modules available separately Description Reference Quantity Chromatin shearing optimization kit Low SDS C01020010 1 kit Chromatin shearing optimization kit Medium SDS C01020011 1 kit Chromatin shearing optimization kit High SDS C01020012 1 kit Auto Pure C02010012 100 rxns Table 4 Plastics and consumables available separately Description Reference Quantity 200 ul tube strips 12 tubes strip cap strips C30020001 80 200 ul tube strips 8 tubes strip cap strips for SX 8G IP Star Compact C30020002 120 96 well microplates for IP Star C30080030 10 Tips box C30040021 960 Tips bulk C30040020 1000 2 ml microt
10. ERICA 400 Morris Avenue Suite 101 Denville NJ 07834 USA Tel 1 862 209 4680 Fax 1 862 209 4681 orders naUMdiagenode com For a complete listing of Diagenode s international distributors visit www diagenode com en company distributors php For rest of the world please contact Diagenode s a info na diagenode com info diagenode com O 2014 Diagenode Inc All rights reserved The content of this document cannot be reproduced without prior permission of the authors Bioruptor and IP Star are registered trademarks of Diagenode
11. IB 77 pal 98 ul 2 Empty Empty 3 Magnetic beads 10 pl 20 50 ul Magnetic beads 10 ul 20 50 ul h ChIP Buffer H 30 ul 100 ul ChIP Buffer H 90 ul 100 pl 5 ChIP Buffer H 90 pl 100 pl ChIP Buffer H 90 ul 100 pl 6 ChIP Buffer H Ab 100 pl 100 pl ChIP Buffer H Ab 100 pl 100 pl 7 Sheared Chromatin Mix 100 pl 200 pl Sheared Chromatin Mix 100 pl 200 pl 8 Wash Buffer H1 100 pl 150 pl Wash Buffer H1 100 pl 150 pl 9 Wash Buffer H2 100 pl 150 pl Wash Buffer H2 100 pl 150 pl 10 Wash Buffer H3 100 pl 150 pl Wash Buffer H3 100 pl 150 pl 11 Wash Buffer H4 100 pl 150 pl Wash Buffer H4 100 pl 150 pl 12 Elution buffer H 5M NaCl 96 pl 4 pl 96 ul 4 ul DIB 100 pl 100 pl This Auto Histone ChIP seg kit has been optimized with Diagenode s high quality ChlP grade antibodies and we use very low amounts of antibody per IP The binding capacity of 10 ul of magnetic beads is 3 ug of antibody If you plan to use more than 3 ug of antibody per IP we recommend that the quantity of beads Is adjusted accordingly Please contact us for advice If required NOTE 1 If required add 2 ul of BSA 5 to the sheared chromatin mix when running ChIP 100 ul protocols or 4 ul of BSA 5 when running ChIP 200 ul protocols NOTE 2 If required NaBu HDAC inhibitior 20mM final concentration or other inhibitors can also be added to the sheared chromatin mix Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Be
12. IENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 9 Kit Materials Kit Content The Auto Histone ChIP seq kits contains reagents to perform 16 or 100 Chromatin Immunoprecipitations by using the SX 86 P Star Automated System The kit content is described in Table 1 Upon receipt store the components at the temperatures indicated in Table 1 Note The kit has been designed to work with histone antibodies and allows to perform ChIP experiments starting with sheared chromatin Table 1 Kit content Description Quantity x16 Quantity x100 Storage 1 25 M Glycine 2 ml 115 mil 4 C Protein G or Protein A coated magnetic beads 220 ul 1500 ul 4 C ChIP Buffer H 20 ml 125 ml 4 C Rabbit IgG or Mouse IgG 1a 110 ul 4 C Protease inhibitor mix 200x 100 ul 700 ul 20 C 5 BSA 10x solution 40 ul 200 ul 20 C 1 M Sodium Butyrate 40 ul 200 ul 20 C Wash Buffer H1 4ml 30 ml 4 C Wash Buffer H2 4ml 30 ml 4 C Wash Buffer H3 4 ml 30 ml AG Wash Buffer H4 4 ml 30 ml 4 C Elution Buffer H 4ml 30 ml AG 5 M NaCl 220 ul 1500 ul 4 C Water 2ml 10 ml LC DNA Isolation Buffer DIB 4ml 30 ml 4 C Proteinase K 40 pl 220 ul 200 Table 2 Reagents available separately Description Reference Description Quantity Storage 1 M Sodium butyrate 120
13. NODE customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at DIAGENODE We therefore encourage you to contact us If you have any suggestions about product performance or new applications and techniques For technical assistance and more information call the DIAGENODE Technical Service Department or contact your local distributor Diagenode s a BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA LIEGE SCIENCE PARK Rue Bois Saint Jean 3 400 Morris Avenue Suite 101 Denville NJ 07834 USA Tel 1 862 209 4680 Fax 1 862 209 4681 techsupport na diagenode com 4102 Seraing Belgium Tel 32 4 364 20 50 Fax 32 4 364 20 51 techsupport diagenode com orders na diagenode com orders diagenode com For a complete listing of Diagenode s international distributors visit http www diagenode com en company distributors php For the rest of the world please contact Diagenode s a Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com Ordering information Description Cat No NEW Cat No OLD Format SX 8G IP Star Compact B03000002 UH 002 0001 1 unit Auto True MicroChIP kit C01010140 16 rxns Auto True MicroChIP amp MicroPlex Library
14. Prep Package CO1010141 A Te eee aa prep rxns MicroPlex Library Preparation kit x12 C05010010 AB 004 0012 12 rxns Auto Histone ChIP seg kit protein A x16 C01010020 AB Auto02 A016 16 rxns Auto Histone ChIP seg kit protein A x100 C01010022 AB Auto02 A100 100 rxns Auto Histone ChIP seg kit prowwtein G x16 C01010021 AB Auto02 G016 16 rxns Auto Histone ChIP seg kit protein G x100 C01010023 AB Auto02 G100 100 rxns Auto Transcription ChIP kit protein A x16 C01010030 AB Auto03 A016 16 rxns Auto Transcription ChIP kit protein A x100 C01010032 AB Auto03 A100 100 rxns Auto Transcription ChIP kit protein G x16 C01010031 AB Auto03 G016 16 rxns Auto Transcription ChIP kit protein G x100 C01010033 AB Auto03 G100 100 rxns Auto ChIP kit protein A x100 C01010011 AB Auto01 A100 100 rxns Auto ChIP kit protein G x100 C01010013 AB Auto01 G100 100 rxns Auto MeDIP kit x16 C02010011 AF Auto01 0016 16 rxns Auto MeDIP kit x100 C02010012 AF Auto01 0100 100 rxns Auto hMeDIP kit x16 C02010033 AF Auto02 0016 16 rxns Auto MethylCap x48 C02020011 AF Auto01 0048 48 rxns Auto Pure kit C03010010 AL Auto01 0100 100 rxns Visit us at one of Diagenode s demo sites or discover our Automated Systems by performing some assays with the help of our R amp D and Technical Department Diagenode s a BELGIUM EUROPE LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Seraing Belgium Tel 32 4 364 20 50 Fax 32 4 364 20 51 ordersOdiagenode com Diagenode Inc USA NORTH AM
15. RECT ChIP PCR tube 100000000 100000000 Semple 200000000 800000000 Well 6 Ab in buffer 700000000 100 ul 6500000000 Well 3 Magnetic beads 500000000 00000000 200000000 Well 7 Sample IP wash 3 Wash Buffer H3 IP wash 4 Wash Buffer H4 INDIRECT ChIP PCR tube Well 7 Sample Ab 10041 Well 3 Magnetic beads UY vue NOTE 1 If required add 2 ul of BSA 5 to the chromatin sample when running ChIP 100 ul protocols or 4 ul of BSA 5 to the chromatin sample when running ChIP 200 ul protocols NOTE 2 If required NaBu HDAC inhibitior 20mM final concentration or other inhibitors can also be added to the chromatin sample Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 17 DIRECT ChIP INDIRECT ChIP Set confirmation Set confirmation Protocol and Sample Protocol and Sample Protocol Protocol Sample number Sample number Configuration Configuration Ab coating h IP reaction IP reaction h Beads incubation Washes min Washes Current temperature Current temperature Left block C Right block Left block C Right block Back diagenode diagengHe Screen Set confirmation After the user presses the next button in the Layout information screen the Set confirmation screen appears At this point user is expe
16. analysis methods meetings workshops and services Involved e Validation of your primers Test primer sets by in silico PCR http genome cse ucsc edu cgi bin hgPcr Primers should amplify unique DNA products from the genome Test every primer set in qPCR using 10 fold serial dilutions of input DNA Calculate amplification efficiency AE of primer set using the following by formulal5 AE 10 1 slope The ideal amplification factor Is 2 If itis not the case the gPCR reagents from different brand or new primers should be tested QPCR products should also be run on a high resolution agarose gel since melting curve analysis in qPCR not always picks up primer dimmer or additional products Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 29 Data interpretation The efficiency of chromatin immunoprecipitation of particular genomic locus can be calculated from qPCR data and reported as a percentage of starting material ChIP Total input ChIP Total input 2 Ctlx input loglx log2 Ct ChIP x 100 Here 2 is the amplification efficiency AE as calculated abovel5 Ct ChIP and Ct x input are threshold values obtained from exponential phase of qPCR for the IP d DNA sample and input sample respectively the compensatory factor logx lo
17. ch reverse and forward 12 5 ul of master mix e g iQ SYBR Green supermix 5 0 ul of purified DNA sample and diluted purified inputls see above for input dilution 6 9 pl of water qPCR cycles Temperature Time PCR Amplification 7a E 3 minutes x1 ome 30 seconds 60 C 30 seconds x40 120 30 seconds Melting Curve 69 C and increment of 0 5 C per cycle 1 minute x60 2 When the PCR is done analyse the results Some major advices are given below e Your own primer design Self complementarity and secondary structure of the primers can be tested for primer design http frodo wi mit edu cgi bin primer3 primer3_www cgi Annealing temperature of 60 C is recommended for gPCR primers Short length of amplified DNA fragment 50 150 bp facilitates the PCR efficiency and reduces potential problems in amplification of G C rich regions Difference in melting temperature between forward and reverse primers should not exceed 2 to 3 C G C stretches at the 3 end of the primers should be avoided e Advantages of the qPCR GPCR or Real time PCR enable fast quantitative and reliable results Visit http www gene quantification info The Gene Quantification page describes and summarises all technical aspects Involved in quantitative gene expression analysis using real time qPCR amp qRT PCR It presents a lot of applications chemistries methods algorithms cyclers kits dyes
18. cted to be ready to press RUN Buttons e The user presses the Back button The user returns to the Layout information screen e The user presses the Run button This is the expected action when user gets to this display after reviewing blocks Runs the protocol Running status Protocol name Progress Bar Remaining time Remaining time Left block L0 E Right olock diagenctie Current Temperature Value Screen Running After the user presses the Run button in the Set confirmation screen the Running screen appears Buttons e The user presses the Stop button Then screen shall be changes to Stop Dialog Status screen is preferred as a progress bar that moves across the screen as the step progresses Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 18 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Running status 4 Remaining time A ChIP DIB CAUTION e Add INPUT sample in well 1 Add Proteinase K 1 ul in well 1 and 12 e And put the cap on the tubes diagencde Screen Elution INPUT is defined as a 100ul protocol INPUT 1 pul diluted sheared chromatin 99 ul DIB buffer b 200 ul protocol INPUT 2ul diluted sheared chromatin 98 ul DIB Buffer Running status 2 Remaining time Left block diagenotie Finish e Open the door e Recover the samples and remove the consumables 4 300000000
19. ction including antibodies and buffers and also provides you with fewer buffers in comparison with other kits The Auto Histone ChlP seq Kit has been validated to perform ChIP seg experiments using antibodies directed against histone modifications The combination of this high quality kit and the IP Star allows Chromatin IP to be performed in less than 10 hours Starting with sheared chromatin the Automated System provides purified immunoprecipitated DNA from your sample The Auto ChIP kit protocol has been validated using chromatin sheared by sonication using the Bioruptor Not only does the IP Star eliminate the problem of human variation associated with producing our samples it also enables us to produce 1000 2000 ChlP seq samples per year very reliably SE The P Star reduces our processing time down from one day of manual work to just one overnight Feedback run with only 30 minutes of hands on work The IP Star has made all our ChIPs consistent and the process completely reliable regardless of the operator or the time of day Dr John Lambourne Postdoctorate Researcher at the Innovation Centre McGill University Canada Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 5 SX 8G IP Star and SX 8G IP Star Compact Systems for automation of epigenetic applications Diag
20. day s high resolution epigenomic studies Diagenode Automated Platforms replace the numerous manual error prone steps of complex epigenetic applications with a reliable highly consistent and automated process that requires minimal operator intervention We empower researchers to simplify the tedious protocols and the complexity of many epigenetic protocols In addition Diagenode Automated Systems minimize sample carryover data variability and costly errors The platforms offer full workflow Support for epigenetics research utilizing our complete kits and laboratory validated protocols to rapidly deliver high quality and consistent data Auto Histone ChIP seq kit The Auto Histone ChIP seq kit was developed to enhance the utility of the ChIP procedure allowing one to perform many more ChlPs per day and per week The entire procedure can be performed in a single day since two overnight incubations have been eliminated The IP has been optimized to specifically select and precipitate the chromatin with the use of our validated antibodies buffers and protocols Furthermore the use of our automated system will drastically increase the consistency of your ChIP assay The Auto Histone ChIP seq kit allows quick and highly specific chromatin IP sample analysis The Auto ChIP kit protocol has been improved to allow researchers to work with smaller volumes than other traditionally used methods The kit ensures the use of small amounts of reagents per rea
21. diagengdte Innovating Epigenetic Solutions AUTO HISTONE ChIP seg KIT Auto Histone ChIP seg kit protein A x16 Cat No C01010020 Old Cat No AB Auto02 A016 Auto Histone ChlP seq kit protein G x16 Cat No C01010021 Old Cat No AB Auto02 G016 Auto Histone ChIP seqg kit protein A x100 Cat No CO1010022 Old Cat No AB Auto02 A100 Auto Histone ChlP seq kit protein G x100 Cat No CO01010023 Old Cat No AB Auto02 G100 Version 5 21 01 14 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 3 Contents Ligas Us A A II esi aes ek Aenea II ke eae as oe 4 SX 8G IP Star Automated System for ChIP MeDIP amp MBD 1 ees 5 it MOOG ONCEVICW g2uchye een etuens ries et ek set ee io ee eee 8 SUE i ee ee ee ee ee ee ee ee ee ee ee eee ees eee eee 9 LA III EE E EE E E EE E E El Required Materials Not provided sa acoso stare dore pradera dopaje due atea te eee do qr ate da da ote 10 How to perform Automated ChIP in the SX 8G IP Star Compact o ccoccccccc ee 11 ENTMad prepara on Gracey danes nal ae ee ee oe rre die de ee eb eee ad lores 12 PONOT el POC ee O po edna age ee pane web a we Bare ce er ene ep re eS tase Gear wg ee ee we A 13 How to perform Automated ChIP in the SX 8G IP Star eee 20 Choma Phe varatOn avere tages de eed wee desa ode Bud ode bea ea da epale odes 21 eo eo A ee to pee eee eb a pHa ec e d ee eased A oe oe een euae ear 22 PO Gra TV CG hs az hcg ak ood O euree Bee ee geeks ele o au a
22. enode has developed two automated platforms SX 8G IP Star and SX 8G IP Star Compact designed to increase your labs productivity efficiency and experimental reproducibility The two automated platforms are capable of processing up to 16 samples per cycle The automated systems processes sheared chromatin lor DNA to deliver purified DNA ready for qPCR amplification microarray and sequencing analysis Both the SX 8G IP Star and SX 8G IP star Compact have an easy to use open software that provides you with flexibility This allows you to create your personal protocol according to your specific needs Major benefits of Diagenode Automated Platforms SX 8G IP Star Compact Sx 8G IP Star 7 High resolution ChIP seq and MeDIP seq profiles Automated library preparation for Next Generation sequencing Reduces hands on time to just 30 minutes Reduces variability between operators and labs Ideal for low sample starting amounts E a 7 s a e Compatible with Diagenode Kits Auto ChIP kit Auto Histone ChIP seq kit Auto Histone ChIP seq kit Auto MeDIP kit Auto MethylCap kit Auto hMeDIP Auto Pure kit Reduces cross contamination y Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 6 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL ss SX 8G IP Star Compact SX 8G IP Star ChIP seq MeDIP seq MethylCap seg hMeDIP IPure Sample preparation Re ChIP MagB
23. er IP Reagents and sheared chromatin were identical per assay The standard deviations between the four ChlPs performed by the SX 8G P Star are displayed PAGE 7 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 8 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Kit Method Overview Chromatin DNA Shearing Chromatin DNA Preparation Bioruptor Sonication Increased Reproducibility m Automated amp High Throughput m No Foaming E m Chromatin Shearing Optimization kit Low SDS Medium SDS and High SDS No Risk of Contamination ab A O Next Gen Sequencing GAPDH Bioruptor Pico ah ENRICHED Magnetic IP Size Selection re y Auto MethylCap Kit E Library Preparation E lumina TruSeq ChIP miPurekt 0 m NEBNext ChIP seq magnetic purification mw DNA Isolation Buffer a 4 l DNA Purification m MicroPlex Library Preparation kit 50 pg multiplex manual Figure 3 Diagenode provides a full suite of automated solutions for ChIP experiments For Step 1 we offer products to isolate nuclei and chromatin Step 2 describes reproducible sample shearing with the Bioruptor product line In Step 3 and Step 4 the Diagenode IP Star Compact provides error free walk away automation for all your immunoprecipitation and antibody capture needs Europe Diagenode sa LIEGE SC
24. g2 is used to take into account the dilution 1 x of the input The recovery is the ChIP Total input Or input AE Ctinput CtChIP x Fd x 100 Here AE is amplification efficiency as calculated above 5 CtChIP and Ctinput are threshold values obtained from exponential phase of qPCR Fd is a dilution factor of the input DNA to balance the difference in amounts of ChIP and input DNA taken for gPCR Relative occupancy Relative occupancy can be calculated as a ratio of specific signal over background Relative occupancy can be calculated as a ratio of specific signal over background Occupancy input specific loci input background loci Relative occupancy is then used as a measure of the protein association with a specific locus it provides clues about specificity of ChIP Highly specific ChIP can result in about 10 fold enrichment over background and some antibodies can reach up to 1000 fold This value not only depends on the antibody but also on the target ChIP result can be considered as reliable in case of significant values for both efficiency and specificity Use of a standard curve generated from fragmented genomic DNA A dilution series is made and qPCR is run on DNA with the primer one uses for ChIP This will give the PCR efficiency Most qPCR programs allow automatic calculation of the DNA quantity in the samples by comparing with the Ct and known quantities of DNA standards Diagenode Inc North Amer
25. genode magnetic beads BSA is however provided in this kit for customers that would like to proceed with preblocking or to add BSA in the immunoprecipitation reaction For preblocking proceed as follow 1 Pippette 100 ul of beads Add 390 ul CHIP Buffer H and 10 ul BSA 5 0 1 final concentration 2 Incubate 60 minutes by rotation 3 Washed once with 100 ul of ChIP BUffer H 4 Resuspend your beads in 100 ul ChIP Buffer H 10 ul of magnetic beads will bind up to 2 5 ug of Antibody Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com Running a protocol Automated Epigenetic Applications on Magtration Technology Genetein software ver 1 0 Copyright 2010 diagengde Protocols Maintenance Information diagengde Protocols de gt he A A a e hMeDIP MethylCap gt lt OL a MagBisulfite RNA IP diagend e ChIP preparation method 1 Direct method Ab Beads Ag Beads pre wash 5 Antibody coating l 3 IP reaction 5 4 IP washes 5 Elution 2 Indirect method Ag Ab Beads Beads pre wash IP reaction 3 Beads incubation 4 IP washes 5 Elution diagenotie DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 13 Diagenode Splash Screen A0 After the software start up screen disappears the Diagenode splash screen Is displayed for several seconds and then disappear
26. hyde per 500 ul sample Mix by gentle vortexing and incubate for 8 minutes at room temperature to allow fixation to take place 4 Stop the fixation by adding 97 ul of Glycine solution Mix by gentle vortexing and Incubate for 5 minutes at room temperature Work on ice from this point onwards 5 Centrifuge at 1 600 rpm 250 x g for 5 minutes at 4 C and gently aspirate the supernatant without disturbing the cell pellet 6 Wash the cells twice with 1 ml PBS STEP 2 Cell lysis and chromatin shearing We recommend the use of Diagenode Bioruptor devices in combination with our shearing optimization kits Table 3 p 10 for the preparation of the sheared chromatin STEP 3 Sheared chromatin and beads preparation 1 Start with sheared chromatin in Low Medium or High SDS concentration depending on the cell type and the amount of cells sheared 2 Dilute your sheared chromatin with ChIP Buffer H to reach a final concentration of 0 1 0 15 SDS Dilute 10 times if using Chromatin shearing optimization kit High SDS and 5 times if using Chromatin shearing optimization kit Medium SDS 3 Protease Inhibitors are provided in the kit Add to the diluted sheared chromatin protease Inhibitors to a final concentration of 1X Note 1 please mind in advance about the cell concentration during shearing process as the working IP volumes in the automated systems are 100 ul and 200 ul Note 2 In general Diagenode does not recommend to preblocked Dia
27. ica Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 30 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Results Pri Browser on Human Mar 2006 NCBI36 hg18 Assembly SIDE aonn 15 E ama 1 EJE Sca le 26 kb cweig esssceco escssace eEssoscec Eessoeeae ezc ousoo szsosepo ESdieees ssqieopal esqco0oB essasoool Histone nodifications 25 TCOB1CD4Na iwe_H3K3SSmes3 TC601CD4MNa ive H3 H3K36me3 apie nn we Fae de e adaha e ed is ml ole TCOBLCOSNS iYe JEK4NeS TC68 1 CDENA ve _H3x H3K4meds pA ITP Te Arranca TTT Tt ee a TCO I COBYA Wve MIH 1 hk H3K9me3 TCOBLCOSNS ive HEK4net TOG601C0DENA ivo H3 H3K4me1 j oe mdo a Aa b pado JA de e dl Esti vs dh ima Ll art A PUT A eee bd ud desis td im a dell ll lodos al TIE AT y hiabbidid l astcdets tve NSkSMic TC 1CDENa ive HSK H3K2 me3 TCO 1 CDEN S ive Ha H3K9Ac 1 hsm h aa lll al ob a LM dinl hiaan laina b ha la Laa La Ll daniu LA ka TON Genes Enc d on RefSeq UniProt TGenkank eros ahd Comparative Genomics aFe 4 m4 o o o NF 274 4 4 _ ZhF274 mp o mo cnf 2 4 Bebe ooo ee ee Oe oe ee eee ee eee Figure Auto ChlP seq assays were performed on the SX8G P Star using primary human CD4 and CD8 T cells isolated by negative magnetic separation of peripheral blood mononuclear cells Each Auto ChIP sample was performed using Diagenodes Auto Hist
28. in in both protocols 1 Switch on the SX 8G IP Star The power switch is on the right side of the instrument 2 Switch on the computer 3 Start SX 86 V32 software through the following icon LJ 4 Place the prepared tube strip on the right cooling heating block of the workstation sis Se CAE AS aoe 0000 001 ET AA 000000000000 A 0640000000000 0000 F HEPATITIS AAA 7 i Bp pp pp p a 2228 pp p a PERE 9 Close the workstation door and lock it using the following icon fy Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 25 6 Press the following icon E Select the protocol of interest Press start X8G V5 Ver0 7 ChIP_Small_Demo HLD MN The program ended successfully diagengy IMPORTANT NOTE If the ChIP protocols do not appear in the screen 1 Open the SX 8V52 directory 2 Open Easy start ini file Write the directory location of the protocols The Easy start ini file should contain the following information EASYSTARTSCREEN HoldFilePath C Documents and Settings Desktop New software protocols ChIP Ab Coating for loading ChIP Direct protocols or HoldFilePath C Documents and Settings Desktop New software protocols ChIP IP and beads incubation for loading ChIP Indirect protocols In red it is indicated the
29. indow will be displayed indicating the step that the protocol is processing 1 Add 1 Input to well 1 1 ul of input when using the 100 ul protocol 2 ul of input using the 200 ul protocol 2 Close the tube strip with the corresponding caps 3 Press OK 4 Reverse crosslinking will be performed at 65 C for 4 hours or O N pes 1 input Fe fp s eli7i s gt o n Note Optional RNase treatment by incubating the samples with RNase at 37 C during 30 minutes can be performed after the reverse crosslinking Diagenode does not provide RNase 9 DNA purification can be done using our simplified and validated Auto IPure kit from Diagenode Alternatively spin columns and Phenol chloroform method can also be used 9 Shutting down the IP Star 1 Click on File and press End to close the software correctly 2 Switch off the computer and its monitor 3 Switch off the SX 8G IP Star Automated System power switch on the right side Note Ensure that the door is closed PAGE 27 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 28 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Quantitative PCR amp Data Analysis This last step consists in amplifying and analysing the IP d DNA 1 Prepare the qPCR mix using SYBR PCR Green master mix qPCR cycles are given below qPCR mix total volume of 25 ul reaction 1 ul of provided primer pair stock 5 uM ea
30. isulfite RNA IP Library preparation for NGS platforms ChIP seq MeDIP seq MethylCap seq hMeDIP lPure Sample preparation Re ChIP MagBisulfite RNA IP Applications Protocols Sample prep MeDIP hMeDIP MethylCap e gt A po A Software EAW R he ChIP Re ChIP MagBisulfite RNA IP lt E 4 yy IPure Library prep diagencde User interface Intuitive touch screen panel PC Software User friendly Software training not required Software training before use Dispensing Automated dispension of assay reagents Manual dispension of assay reagents Antibody coating temperature time mixing Protocol rater speed optimization ae l Antibody coating ltemperature time Immunoprecipitation temperature time A flexible H Immunoprecipitation temperature time mixing speed parameters Washes temperature time mixing speed 750W x 740 D x 610 H 100 kg 1070W x 650 D x 780 H 130 kg Characteristics Noria 20690 8 Nozzles X Y Z axis 4 95 C Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com Improved reproducibility DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Our SX 8G P Star will increase the immunoprecipitation reproducibility between IPs performed by the same as well as by different operators see figure 1 and 2 below Reagents Antibodies buffers a and sheared chromatin were iden
31. lgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL ChIP Indirect method IP and beads incubation With this method the antibodies are incubated first with the sheared chromatin and after that the magnetic beads are added to the immunocomplex m Elution buffer Input or DIB i Chromatin Ab Bead washes Wash y eens Elution buffer IP or DIB 1 2 3 4 5116 7 8 9 10 1 12 gt gt gt gt gt gt lt gt lt gt lt gt lt lt A IPURE Psa seh E on eae lee 1 Elution buffer H 5M NaCl 95 ul 4ul 94ul4 4yul DIB 99 ul 98 ul 2 Empty Empty 3 Magnetic beads 10 ul 20 50 ul Magnetic beads 10 ul 20 50 ul 4 ChIP Buffer H 50 pl 100 pl ChIP Buffer H 90 ul 100 pl 5 ChIP Buffer H 50 pl 100 pl ChIP Buffer H 90 pl 100 pl 6 ChIP Buffer H 100 pl 100 pl ChIP Buffer H 100 pl 100 pl 7 Sheared Chromatin Mix Ab 100 pl 200 pl Sheared Chromatin Mix Ab 100 pl 200 pl 8 Wash Buffer H1 100 pl 150 pl Wash Buffer H1 100 pl 150 ul 9 Wash Buffer H2 100 pl 150 pl Wash Buffer H2 100 pl 150 pl 10 Wash Buffer H3 100 pl 150 pl Wash Buffer H3 100 pl 150 pl 11 Wash Buffer H4 100 pl 150 pl Wash Buffer H4 100 pl 150 pl 12 Elution buffer H 9M NaCl 96pl 4pl 96pl 4pl DIB 100 pl 100 pl This Auto Histone ChIP seq kit has been optimized with Diagenode s high quality ChlP grade antibodies and we use very low amounts
32. n DIB_1 day File Name Status PLG0 C iDocuments and Settings igniacio My Completed A 1 CiDocuments and SettingsilgniaciolMy WakeUp B fee C D E F G H Lig_in POP P_SPEED_H Asp Speed wait msec 200 waittime msec Stack DUP Lig_out POP P_SPEED_H Disp Speed Pass_time IF_Goto LE 7200 Mixing Time Sec Repeat Stack Drop 8 Reverse crosslinking F Simul 1 9 2 Volume Mixing 1 9 3 Volume Mixing 1 9 4 Volume Mixing 1 9 5 Air Dsp 1 9 6 Magnet OFF O 1 9 7 Interium Height Z Move 1 10 1 Right block vVell5 1 10 2 Action Z 1 10 3 Pre asp 1 10 4 Beads resuspend 1 10 5 Pre Dsp 1 11 2 Volume Mixing 1 11 3 Magnet ON 1 11 4 Volume Mixing 1 11 5 Air disp 1 11 6 Magnet OFF O 1 11 7 Air Dsp 1 11 8 Interium Height Z Move 1 12 1 0 1 12 2 IP reaction O 1 1 2 3 J0 1 13 1 Right block vVell6 1 13 2 Action Z 1 13 3 Beads resuspend 1 13 4 IP reaction O 1 13 5 Air Dsp 1 13 6 Interium Height Z Move 1 13 7 Tip Discard 1 14 1 New Tip Collect 1 14 2 Right block vVell6 1 14 3 Action Z 1 14 4 Beads resuspend After the IP washes the following window will be appear Attention Please OP sass CAUTION gt gt gt gt ES Open the door 4dd 114 input in well 1 And put the cap on the PCR tube During protocol the next w
33. o 10 million cells in 500 ul of PBS Aliquot 500 ul of cell suspension in 1 9 ml tubes Add 13 5 ul of 36 5 formaldehyde per 500 ul sample Mix by gentle vortexing and incubate for 8 minutes at room temperature to allow fixation to take place Stop the fixation by adding 97 ul of Glycine solution Mix by gentle vortexing and incubate for 5 minutes at room temperature Work on ice from this point onwards Centrifuge at 1 600 rpm 250 x g for 5 minutes at 4 C and gently aspirate the supernatant without disturbing the cell pellet Wash the cells twice with 1 ml PBS STEP 2 Cell lysis and chromatin shearing We recommend the use of Diagenode Bioruptor devices in combination with our shearing optimization kits Table 3 p 10 for the preparation of the sheared chromatin STEP 3 Sheared chromatin and beads preparation 1 Start with sheared chromatin in Low Medium or High SDS concentration depending on the cell type and the amount of cells sheared Dilute your sheared chromatin with ChIP Buffer H to reach a final concentration of 0 1 0 15 SDS Dilute 10 times if using Chromatin shearing optimization kit High SDS and 5 times if using Chromatin shearing optimization kit Medium SDS Protease Inhibitors are provided in the kit Add to the diluted sheared chromatin protease inhibitors to a final concentration of 1X Note 1 please mind in advance about the cell concentration during shearing process as the working IP volumes
34. one ChIP seq kit reagents and contained 1 ug of input chromatin Illumina Genome Analyzer libraries were prepared and the samples were sequenced at the DNA Technologies Core at UC Davis Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 31 Aditional Protocols Sheared chromatin analysis This protocol refers to the Diagenode s Elution module Cat No mc magme 002 that can be ordered separately Reagents not supplied 9 e RNase cocktail e g Ambion AM 2286 A e Phenol chloroform isoamyl alcohol 25 24 1 e Chloroform isoamyl alcohol 24 1 e 100 Ethanol e 70 Ethanol e Agarose and TAE buffer e TE Take an aliquot of 100 ul of sheared chromatin and spin the chromatin at 14 000 x g 13 000 rpm for 10 min at 4 C Transfer the supernatant to a new tube for chromatin analysis A minimum of 60 000 cells is needed to be vizualized onto agarose gel If each 100 ul of sheared chromatin correspond to 10 000 cells then perform 6 reactions in parallel and pool the DNA pellets obtained at Step 14 during respuspension in TE Prepare RNase cocktail dilution e g Ambion AM 2286 A dilute 1 ul of cocktail in 150 ul of water Add 2 ul of diluted RNase cocktail to the chromatin Incubate 1h at 37 C Prepare the Complete Elution Buffer by mixing thoroughly Buffer D E
35. rotocol peltier block screen B to eliminate possible condensation in the block diagencde NO YES CAUTION Temperature unit maintenance is Defined protocol name lists executed diagencte CAUTION Temperature unit maintenance x Remaining time S Right block diagencde Note 1 Please note that when isolating DNA with DIB buffer the DNA will be recovered in single strand conformation When isolating DNA with Elution Buffer followed by reverse crosslinking the DNA will be recovered in double strand conformation Note 2 RNase treatment by incubating the samples with RNase at 37 C during 30 minutes can be performed after the reverse crosslinking and it is recommended for Chlp seq experiments However Diagenode does not provide RNase Note 3 DNA purification can be done using our simplified and validated Auto Pure kit from Diagenode Alternatively spin columns and Phenol chloroform method can also be used Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com How to perform Automated ChIP in the SX 86 IP Star SX 8G IP STAR DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 21 How to perform Automated ChIP in the SX 8G IP Star Chromatin preparation STEP 1 Cell collection and DNA protein crosslinking 1 Z Collect the cells by trypsinisation and wash two times with PBS Count the cells and resuspend them in PBS to obtain up t
36. s Start Screen Top menu After the Digenode splash screen disappears the start screen Is displayed This is the first active window It allows the user to enter into three different parts of the software USER ACTIONS Buttons e Protocols e Maintenance for technical service e Information Diagenode contact details Protocols screen All avallable protocols are displayed on this screen Screen ChIP preparation methods The user can select between protocols for direct or indirect ChIP methods Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 14 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Screen Categories Name Protocol List After the user presses the Categories Namel button the Categories Name appears When selected the protocol on the protocol list the Run button shall turn executable Defined protocol name lists Buttons e The user presses the Back button The user returns to the Protocols screen e The user presses the Shutdown button The screen shall be changed to Power Off diagengte e The user presses the Run button The screen shall be changed to Sample number e A Page up the list box e VY Page down the list box Sample number Screen Sample number After the user presses the Run button the Sample number appears Buttons Sample number e The user presses
37. the Sample number Text box Then screen will be changed to keyboard e The user presses the Back button The user returns to the Protocol List screen diagenotie e The user presses the Next button Then screen shall be changed to Configuration or Layout information ne number Keyboard Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL PAGE 15 DIRECT ChIP INDIRECT ChIP Configuration Save Parameter Configuration C Soon Mixing time Temperature Mix Speed Mixing time Temperature Mix Speed Ab coating IP reaction Cc IP reaction C Beads incubation 19 C diagencte M amp diageng e Screen Configuration After the user presses the next button from the Sample number screen the Configuration screen appears Buttons e The user presses the Back button The user returns to the Protocol List screen e The user presses the Next button The screen shall be changed to Layout information Keyboard e The user presses the Save Parameter button The screen will Confirmation be changed to Save Parameter Confirmation OK Current parameters shown in the Display View will Overwrite parameters be stored to the Protocol ptd And returns the user to the display of the Configuration
38. tical for ManChIP and AutoChIP The SX 8G IP Star Automated system removes variation that can be created by manual handling and allows you to optimize and standardize your assay within a lab The SX 8G P Star is designed to improve the accuracy and the reproducibility of any immunoprecipitiation experiment SDIIgG 0 69 Man ChIP SD H3K9me3 23 84 SD IgG 1 4 SD H3K9me3 2 38 c SD IgG 0 17 o uaa Me SDIH3K9me3 1 12 a oe SD IgG 0 09 ade obn IRA 98 62 95 26 SD H3K9me3 0 65 A a D a ChIP 1 ChIP 2 E ee ChIP 1 ChIP 2 2 ChIP 1 ChIP 2 ES 57 83 56 25 60 0 50 70 43 83 44 75 40 0 34 63 20 0 1 96 0 63 1 54 1 42 Auto ChIP SD I9G 0 28 SD H3K9me3 1 6 100 0 90 0 80 0 70 0 ChIP 1 ChIP 2 ChIP 3 ChIP 4 Si 56 25 54 71 57 83 54 34 a 500 o 40 0 se 30 0 20 0 10 0 1 26 1 00 1 45 0 81 IgG H3K9me3 IgG H3K9me3 IgG H3K9me3 IgG H3K9me3 Figure 1 Manual ChIP Four different operators have each performed two ChIP experiments using H3K9me3 antibody on the genomic region SAT2 positive locus 10 000 Hela cells have been used per IP Reagents and sheared chromatin were identical per assay The standard deviations between the ChIPs performed by the same operator and between the four different operators are displayed Figure 2 Automated ChIP Four ChIP experiments using H3K9me3 antibody on the genomic region SAT2 positive locus have been performed by the SX 8G IP Star 10 000 Hela cells have been used p
39. u ee ene ee oo oe 24 Quantitative PCR amp Data analysis 0 0 eens 28 ROS Sua sia iia eee ee en ee ia ed nes ee ee ee Ge ee eg 30 Additonal Protocol 0 n24acne ee cere ne eee sea eae near eeu aedaweeeeare sn aeGhncasemeeses ee net 31 Troubleshooting GUJE sauma tic at ean ee cee og eae a ree oe 33 Technical ASSIStaMCO o2hccanoeeaen testes bOnana treo ia tame unas Gee ass 34 Ordering Information ccc eee eee es Back Cover Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 4 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL Introduction The Diagenode SX 8G IP Star Automated System automates immunoprecipitation and increases reproducibility Diagenode the leading provider of complete solutions for epigenetics research offers a variety of end to end systems to streamline DNA methylation and chromatin immunoprecipitation workflows Central to this full offering is Diagenode s Automated Systems simple yet robust automated bench top instruments that standardize different epigenetic applications i e ChIP MeDIP or MethylCap Diagenode designed these automation systems to make ChIP and DNA methylation studies accessible and reproducible and ensure consistent data in every experiment Diagenode Automated Systems will produce consistent results from any operator regardless of the day the experimental run or the lab Robust and reproducible results is a major goal of to
40. ube for SX 8G IP Star Compact C30010014 100 Large reagent container for SX 8G IP Star Compact C30020004 20 Medium reagent container for SX 8G IP Star Compact C30020003 10 Required Materials Not Provided Reagents e Gloves to wear at all steps e Phosphate buffered saline PBS e Trypsin EDIA e Formaldehyde fresh MolBiol Grade e gPCR reagents e Quant IT dsDNA HS assay kit Invitrogen Equipment and accessories e DiaMag02 magnetic rack Cat No B04000001 e Cell counter e BioruptorR sonication apparatus e Diagenode 1 5 ml TPX microtubes optimized for chromatin shearing with Bioruptor Cat No ZC30010003 ZC30010004 e Centrifuge for 1 5 ml tube e Vortex e Qubit system e gPCR cycler Europe Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Ougr e Seraing Belgium Phone 32 4 364 20 50 Mail info diagenode com How to perform Automated ChIP in the SX 8G IP Star Compact SX 8G IP STAR COMPACT PAGE 12 DIAGENODE AUTO HISTONE ChIP KIT USER MANUAL How to perform Automated ChIP in the SX 8G IP Star Compact Chromatin preparation STEP 1 Cell collection and DNA protein crosslinking 1 Collect the cells by trypsinisation and wash two times with PBS 2 Count the cells and resuspend them in PBS to obtain up to 10 million cells in 500 ul of PBS Aliquot 500 ul of cell suspension in 1 9 ml tubes 3 Add 13 5 pl of 36 5 formalde
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