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AUTO ChIP KIT MANUAL

1. Input If you set diagenctie 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 Screen Elution INPUT is defined as a 100ul protocol INPUT Tul diluted sheared chromatin 99 ul DIB buffer b 200 ul protocol INPUT 2ul diluted sheared chromatin 98 ul DIB Buffer IMPORTANT Please note that DNA Isolation with DIB buffer and proteinase K delivers single strand DNA that can be directly analyzed by gPCR For downstream applications such as sequencing or arrays DNA needs to be purified by phenol chroloform extraction and converted to double stranded DNA 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 CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 27 CAUTION Screen Caution When
2. Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 33 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 34 DIAGENODE AUTO ChIP KIT USER MANUAL Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman 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 Prep Package C01010141 A Te eee aa prep rxns MicroPlex Library Preparation kit x12 C05010010 AB 004 0012 12 rxns Auto Histone ChIP seq 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 seq kit prowwtein G x16 C01010021 AB Auto02 G016 16 rxns Auto Histone ChIP seq kit protein G x100 C01010023 AB Auto02 6100 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 C016 16 rxns Auto Transcription ChIP
3. 150 ul 9 Buffer A 100 ul 150 ul 10 Buffer A 100 ul 150 ul 11 Buffer C 100 ul 150 ul 12 DIB 100 ul 100 ul This Auto ChIP kit has been optimized with Diagenode s high quality ChIP 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 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 14 DIAGENODE AUTO 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 DIB buffer Input r Chromatin Ab Bead washes Washes DIB buffer IP 100 ul 200 ul Description P protocol protocol 1 OB 99 ul 98 ul 2 Empty 3 Magnetic beads 10 ul 20 30 pil 4 Buffer A 50 pl 100 pl 5 Buffer 50 pl 100 ul 6 Buffer A 100 pl 100 pl 7 Sheared Chromatin Mix Ab 100 pl 200 pl 8 Buffer A 100 pl 150 pl 9 Buffer A 100 pl 150 pl 10 Buffer A 100 ul 150 ul 11 Buffer C 100 ul 150 ul DIB This Auto ChIP kit has been optimized with Diagenode s high quality ChIP grade antibodies and we use very low amounts of antibody pe
4. 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 per IP Reagents and sheared chromatin were identical per assay The standard deviations between the four ChIPs performed by the SX 8G IP 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 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 d Qi A o Next Gen Sequencing GAPDH Bioruptor Pico pu ENRICHED Magnetic IP Size Selection re y Auto MethylCap Kit E Library Preparation B lllumina TruSeq ChIP miPurekt m NEBNext ChIP seq magnetic purification m DNA Isolation Buffer D STEP 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 prod
5. IP Star Compact WA 008 0100 100 Large reagent container for SX 8G IP Star Compact WA 007 0020 20 Medium reagent container for SX 8G IP Star Compact WA 006 0010 10 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com How to perform Automated ChiP in the SX 8G IP Star SX 8G IP STAR PAGE 12 DIAGENODE AUTO ChIP KIT USER MANUAL How to perform Automated ChIP in the SX 8G IP Star A Chromatin preparation STEP 1 Cell collection and DNA protein crosslinking 1 Collect the cells by trypsinisation and wash two times with PBS 2 Countthe cells and resuspend them in PBS to obtain 10 million cells in 500 ul of PBS Aliquot 500 ul of cell suspension in 1 5 ml tubes 3 Add 13 5 ul of formaldehyde Mix by gentle vortexing and incubate for 8 minutes at room temperature to allow fixation to take place 4 Stop the fixation by adding 57 pl 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 1 Start with sheared chromatin in 1 0 596 or 0 1 SDS depending on the cell type and the amount of cells to shear We recommend the use of Diagenod
6. PCR amp Data AnalysiS usse rer EET tner 26 icc a A 28 Troublesnooting GUIAS v xad eau mane ma qul Brad AUR ada a de A der Ae ead end ae ht eae eee 29 TSCHMICAL ASSISTANCE eee 30 Ordering IA TOPMIONONE nd ee Dia ii tries dd edad eds fed ans oe 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 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 today s high resolution epigenomic studies Diagenode Automated Platforms replace the numerous manual error pr
7. Pee Ed E i sossccososos rr rcr TTT rss er ee CER ee E eee ee eee goggogpOgGOOU o oo SA amp RARRARARATA TTIILIILT111 mARARRRT RAT 55 5 PATTI Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 16 DIAGENODE AUTO ChIP KIT USER MANUAL 6 Press the following icon Pi Select the protocol of interest Press start SX8G V52 Ver0 7 ChIiP_Small_Demo HLD 3 RA The program ended successfully diagendod 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 directory location of the ChIP protocols 3 Start now SX 8G V52 software through SX 86 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 Schedule Manager Ver1 0 Title CHP Small Demo Active Blocks 1 Ca Total Process lime Dec Complete Time 1
8. the protocol finishes the user can return to the protocol list screen A or warm the Do you want to start again a protocol peltier block screen B to eliminate possible condensation in the block diagenctie CAUTION Temperature unit maintenance is Defined protocol name lists executed diagenctie diagenctie CAUTION Temperature unit maintenance Remaining time Left block gt Right block E diagenotie Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 28 DIAGENODE AUTO 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 each 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 5 pl of water qPCR cycles Temperature Time PCR Amplification 79 C 3 minutes x1 ome 30 seconds 60 C 30 seconds x40 120 30 seconds Melting Curve 65 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 stru
9. 3 31 C New 5 25 apobcation 1 03 09 SHB Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 17 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 SXKBG Y52 Yer0 7 ChIP_Small_Demo HLD he program ended successfully q diagenctiel Modify Parameter for Chir Save modification protocol Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 18 DIAGENODE AUTO ChIP KIT USER MANUAL 7 The program will run through the following steps magnetic bead washes IP and IP washes Simul 18 2 Volume Ming During protocol the next window will be displayed indicating the 1 9 3 Volume Mixing LES Ar step that the protocol is processing 1 9 6 Magnet OFF 0 1 9 7 Interium Height Z Move 1412 1416 Pitt tty Antibody immobilize to beads gt IP reaction gt DNA purification DIB_1 day File Name Status PLGO CiDocuments and SettingsilgniacioiMy Completed 1 10 1 Right block v ell5 A 1 CiDocuments and SettingsugniacioWy WakeUp 1 10 2 Action Z B si 1 10 3 Pre a
10. AL PAGE 5 SX 8G IP Star and SX 8G IP Star Compact Systems for automation of epigenetic applications Diagenode has developed two automated platforms SX 8G IP Star and SX 8G IP Star Compact designed to increase your lab s 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 or 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 86 IP Star Compact SX 86 IP Star e High resolution ChlP 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 E gt E E gt 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 IPure kit Reduces cross contamination y Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL ss SX 8G IP Star Compac
11. IP diagenote ChIP preparation method 1 Direct method Ab Beads Ag Beads pre wash Antibody coating I i 3 IP reaction 4 P washes 5 Elution 2 Indirect method Ag Ab Beads Beads pre wash IP reaction 3 Beads incubation 4 IP washes 5 Elution diagenctie DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 21 Diagenode Splash Screen AO After the software start up screen disappears the Diagenode splash screen is displayed for several seconds and then disappears 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 available 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 22 DIAGENODE AUTO ChIP KIT USER MANUAL Defined protocol name lists diagenctie Sample number Sample number diagenctie id number Keyboard Screen Categories Name Protocol List After the user presses the Categories Namel button the Categories Name appears When sel
12. IPs 220 pl C kch 802 660 detergent and 0 02 sodium azide 600 ul Do not freeze kch 802 150 included 1500 pl Protein G coated magnetic beads kch 818 220 The beads are supplied for 16 IPs 220 pl 4 C kch 818 660 detergent and 0 02 sodium azide 600 pl Do not freeze kch 818 150 included 1500 ul Rabbit IgG kch 504 250 1 ug ul 250 pl 4 C Mouse IgG kch 819 015 1 ug ul 15 ul 4 C Buffer B kch 805 003 Detergent and chelator mix included Antibodies a 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 ChIP KIT USER MANUAL Table 3 Kits and Modules available separately Description Reference Quantity Chromatin shearing optimization kit Low SDS AA 001 0100 1 kit Chromatin shearing optimization kit Medium SDS AA 002 0100 1 kit Chromatin shearing optimization kit High SDS AA 003 0100 1 kit Pure AL 100 0100 100 rxns Auto IPure AL Auto01 0100 100 rxns Table 4 Plastics and consumables available separately Description Reference Quantity 200 ul tube strips 12 tubes strip cap strips WA 001 0080 80 200 ul tube strips 8 tubes strip cap strips for SX 8G IP Star Compact WA 002 0120 120 96 well microplates WA 003 0010 10 Tips box WC 002 0960 960 Tips bulk WC 001 1000 1000 2 ml microtube for SX 8G
13. TO ChIP KIT USER MANUAL Our SX 8G IP 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 Jand sheared chromatin were identical 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 IP Star is designed to improve the accuracy and the reproducibility of any IMmunoprecipitiation experiment Man ChIP SD IgG 0 94 SD H3K9me3 11 36 100 0 A 80 0 ChIP 1 ChIP 2 60 0 50 70 40 0 34 63 70 0 ChIP 1 56 25 1 26 IgG H3K9me3 SD IgG 0 69 SD H3K9me3 23 84 SD lgG 1 4 SD H3K9me3 2 38 C SD IgG 0 1796 SDIH3k9me3 1 12 es ali SD IgG 0 09 78 62 95 26 SD H3K9me3 0 65 ChIP 1 ChIP 2 57 83 56 25 D ChIP 1 ChIP 2 43 83 44 75 SD IgG 0 28 SD H3K9me3 1 6 ChIP 2 ChIP 3 ChIP 4 54 71 57 83 54 34 1 00 1 45 0 81 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
14. cture 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 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 formula 5 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 produc
15. d DNA isolation 1 ul Proteinase k Importan Note Please note that DNA Isolation with DIB buffer and proteinase K delivers single strand DNA that can be directly analyzed by qPCR For downstream applications such as sequencing or arrays DNA needs to be purified by phenol chroloform extraction and converted to double stranded DNA Shutting down the SX 8G 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 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman 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 20 DIAGENODE AUTO ChIP KIT USER MANUAL How to perform Automated ChIP in the SX 8G IP Star Compact Prepare Reagents STEP 1 Cell collection and DNA protein crosslinking 1 2 Collect the cells by trypsinisation and wash two times with PBS Count the cells and resuspend them in PBS to obtain 10 million cells in 500 ul of PBS Aliquot 500 ul of cell suspension in 1 5 ml tubes Add 13 5 ul of formaldehyde Mix by gentle vortexing and incubate for 8 minutes at room temperature to allow fixation to take place Stop the fixation by addin
16. diagendte Innovating Epigenetic Solutions AUTO ChIP KIT MANUAL Auto ChIP kit protein A x100 New Cat No C01010011 Old Cat No AB Auto01 A100 Auto ChIP kit protein G x100 New Cat No C01010013 Old Cat No AB Auto01 G100 Version 8 27 05 13 Technical Assistance amp Ordering Information Diagenode s a BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA Avenue de l h pital 1 400 Morris Avenue Suite 101 Tour GIGA 3rd Floor Denville NJ 07834 USA 4000 Li ge Belgium Tel 1 862 209 4680 Tel 32 4 364 20 50 Fax 1 862 209 4681 Fax 324 304 20051 techsupport na diagenode com 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 support distributors php For the rest of the world please contact Diagenode s a DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 3 Contents Ligas Us pe cC PTT 4 SX 8G IP Star Automated System for ChIP MeDIP amp MBD 5 MES OO OVCRVICW annata beet eee tk set ee eee ee eek 8 duc Sh ck ass Se oe te te eh ts sh eth th ed ete eee Ge ee ee he rm 9 A OO UE E A El How to perform Automated ChIP in the SX 8G IP Star eee 11 Loading and Fund Protocol cues podria dps a dor ch Rcx de Pa de ida dose dar a 14 lao e ls AA PP 17 How to perform Automated ChIP in the SX 8G IP Star Compact cece 18 ls MA 19 Quantitative
17. e Bioruptor devices in combination with our shearing optimization kits for the preparation of the sheared chromatin 2 Dilute your sheared chromatin with ChIP Buffer at least 10 times if shearing was perform in the presence of 1 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 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 13 B Dispense prepared reagents into the corresponding tubes see picture below ChIP Direct Method Ab Coating 2 Preparation Buffer Ab Antibody x ul ChIP Buffer A 100 x Antibody coating DIB buffer Input ChIP reaction Bead washes DIB buffer IP 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 puo m bordo 1 DIB 99 ul 98 ul 2 Empty 3 Magnetic beads 10 pl 20 50 pl 4 Buffer A 50 ul 100 ul 9 Buffer 50 ul 100 ul 6 Buffer A Ab 100 pl 100 ul 7 Sheared Chromatin Mix 100 ul 200 ul 8 Buffer A 100 ul
18. ected the protocol on the protocol list the Run button shall turn executable Buttons e The user presses the Back button The user returns to the Protocols screen e he user presses the Shutdown button The screen shall be changed to Power Off e he user presses the Run button The screen shall be changed to Sample number e A Page up the list box e W Page down the list box Screen Sample number After the user presses the Run button the Sample number appears Buttons e The user presses 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 e he user presses the Next button Then screen shall be changed to Configuration or Layout information Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 23 DIRECT ChIP INDIRECT ChIP Configuration Save Parameter Configuration D soe Mixing time Temperature Mix Speed Mixing time Temperature Mix Speed Ab coating IP reaction Cc IP reaction C Beads incubation GC o diagencte W diagenctie Screen Configuration After the user presses the next button from the Sample number screen the Configuration screen appears Buttons e he user pr
19. en 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 ChIP 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 Buffer A Elution Buffer DIB IP wash 1 Wash Buffer A IP Wash 2 Wash Buffer A IP wash 3 Wash Buffer A IP wash 4 Wash Buffer C INDIRECT ChIP PCR tube Well 7 Sample Ab 10041 Well 3 Magnetic beads UIIUOII e Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL 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 diage
20. esses the Back button The user returns to the Protocol List screen e he user presses the Next button The screen shall be changed to Layout information Keyboard e he 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 screen m No Returns the user to the display of the Configuration 7 screen e The 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 24 DIAGENODE AUTO ChIP KIT USER MANUAL Layout information diagenctie Layout information Tip Rack DN70 Sample number 8 MIMyel be Block Tip o Reagent information A DIB Elution buffer ul B Beads wash buffer ul C F IP wash No 1 4 ul Block Regent Tip Rack DIRECT ChIP 1109000000 1000000000 9060000000 800000000 7000000090 600000000 5000000090 4000000090 200000000 PCR tube Well 7 Sample 100 ul Well 6 Ab in buffer 100 ul Well 3 Magnetic beads Screen Layout Information After the user presses the next button from Sample number screen or Configuration scre
21. g 57 ul of Glycine solution Mix by gentle vortexing and incubate for 9 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 1 Start with sheared chromatin in 1 0 5 or 0 1 SDS depending on the cell type and the amount of cells to shear We recommend the use of Diagenode Bioruptor devices in combination with our shearing optimization kits for the preparation of the sheared chromatin Dilute your sheared chromatin with ChIP Buffer A at least 10 times if shearing was perform in the presence of 1 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 in the automated systems are 100 ul and 200 ul Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman 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 diagenctie Protocols de gt he A MM hMeDIP MethylCap gt lt e X MagBisulfite RNA
22. 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 IPure 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 Avenue de l h pital 1 Tour GIGA 3rd Floor Diagenode Inc USA NORTH AMERICA 400 Morris Avenue Suite 101 Denville NJ 07834 USA Tel 1 862 209 4680 Fax 1 862 209 4681 orders na diagenode 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 4000 Liege Belgium Tel 32 4 364 20 50 Fax 32 4 364 20 51 orders diagenode com info na diagenode com info diagenode com 2013 Diagenode Inc Al 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
23. low amount of reagents per reaction not only antibodies but also buffers and also provides you with fewer buffers in comparison with other kits The Kit also includes a DNA isolation buffer DIB for an extra fast method to purify your IP d material for qPCR analysis Alternatively for other applications e g sequencing linear amplification or microarray Magnetic DNA purification can be automated as alternative Combination of this High Quality Kit and the SX 8G IP Star Automated System will allow you to perform Chromatin IP in less than 10 hours Starting with sheared chromatin the Automated System will provide you with the purified immunoprecipitated DNA of your sample 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 ChIP seq samples per year very reliably SEIS ThelP Star reduces our processing time down from one day of manual work to just one overnight O ESO 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 CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANU
24. node diagenote 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 expected 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 LUC Rigniygolock Current Temperature Value diagenctie Screen Running After the user presses the Run button in the Set confirmation screen the Running screen appears Buttons e he 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 PAGE 25 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 26 DIAGENODE AUTO ChIP KIT USER MANUAL Running status diagenote 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 diagenctie Running status Remaining time 00 25 00 diagenctie Finish e Open the door e Recover the samples and remove the consumables 4 100000000 2
25. ome position run the protocol again Aspirated liquid drips from the disposable Dripping is acceptable when ethanol is being handled For other liquids air is leaking tips from the syringe pumps Grease or replace the O rings If the problem persists contact DIAGENODE Technical Services Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 32 DIAGENODE AUTO ChIP KIT USER MANUAL Technical Assistance 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 DIAGENODE 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 e customersupport diagenode com e customersupport na diagenode com Europe Diagenode sa CHU
26. one 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 ChIP kit The Diagenode Auto ChIP kit is designed to perform automated immunoprecipitation of Chromatin using the SX G8 IP Star Automated System With the Auto ChIP kit the protocol has been improved to enhance the utility of the ChIP procedure allowing you to perform many more ChIPs per day and per week The entire procedure can be performed in a day s work as the two overnight incubations have been eliminated The IP has been optimized to specifically select and precipitate the chromatin by the use of our antibodies buffers and protocols Furthermore the use of our automated system will drastically increase the consistency of your ChIP assay The Auto ChIP kit allows you to perform chromatin IP analysis of your sample in a FAST HIGH and SPECIFIC manner In the Auto ChIP kit the protocol has been improved to allow researchers to work in smaller tubes than traditionally did so far The kit ensures the use of
27. r 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 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail infogdiagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 15 Loading and 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 9h DIB ChIP_22h_DIB Reagent Preparation 1h Th Magnetic Bead Washes Th Th Ab coating 2h oh Immunoselection oh 15h Washes 20min 20min Add reagents 15min 15min DNA isolation 30min 30min Total Time 10h05 min 23h05 min Input required is sheared chromatin ready to ChIP Performed by using DIB DNA Isolation buffer Note Hands on work time is reduced to 1h45 min 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 8G V52 software through the following icon LJ 4 Place the prepared tube strip on the right cooling heating block of the workstation H ESO AE E o od aga tt D DO 6 9 D
28. rs to amplify a region of the c fos promoter cat pp 1004 050 500 and SAT from the IP d DNA were also used Chromatin was sheared from 100 000 cells Per ChIP experiment 10 000 cells equivalent and 1 ug of antibody were used A negative control antibody negative IgG from rabbit 1 ug IP and no antibody control were included in the ChIP assay A Show efficiency in the recovery for the positive locus SAT2 whereas B show recovery for the negative c fos promoter locus DNA has been isolated by using DNA isolation buffer Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 31 Troubleshooting Guide SX 8G IP Star cannot be switched on SX 8G IP Star is not receiving power Check that the power cord is connected to the workstation and to the wall power outlet Computer cannot be switched on Computer is not receiving power Check that the power cord is connected to the computer and to the wall power outlet SX 8G IP Star shows no movement when a SX 8G IP Star is not switched on Check that the SX 8G IP Star is switched on protocol is started SX 8G IP Star shows abnormal movement The pipettor head may have lost its home position In the Software select Manual when a protocol is started Operation Home After confirming that the pipettor head moves to the h
29. sp zc 1 10 4 Beads resuspend po 1 10 5 Pre Dsp E nw d 1 0 6 Antibo td 0 ES S 1 11 1 Action Z 1 11 2 Volume Mixing gloss 1 11 3 Magnet ON H zm 1 11 4 Volume Mixing 1 11 5 Air disp Jez 1 11 6 Magnet OFF 0 1 11 7 ir Dsp 1 11 8 Interium Height Z Move 1 12 1 1 12 2 IP reaction 0 1 12 3 0 Liq in POP P_SPEED_H Asp Speed 1 13 1 Right block Well6 Wait_msec 1200 waittime msec 1 13 2 Action Z Stack DUP 1 13 3 Beads resuspend Liq out POP HP SPEED H Disp Speed 1 13 4 IP reaction 0 Pass time 1 13 5 Air Dsp IF Goto LE 17200 Mixing Time Sec Repeat 1 13 6 Interium Height Z Move Stack Drop 1 13 7 Tip Discard 1 14 1 New Tip Collect 1 14 2 Right block Well6 1 14 3 Action Z 1 14 4 Beads resuspend 8 ChIP DIB After the IP washes the following window will be appear Attention Please x S lt lt lt lt CAUTION gt gt gt gt Open the door Add INPUT samples 1ul in well 1 Add Proteinase K 1ul manually in well 1 and well 12 And put the cap on the PCR tube Follow the next instructions 1 Add 1 of input in well 1 1 ul of input when using the 100 ul protocol or 2 ul input when using the 200 ul protocol 2 Add 1 ul proteinase K to wells 1 and 12 3 Close the tube strip with the corresponding caps 4 Press OK e 1 gl input 1 pl Proteinase k e IP
30. t SX 8G IP Star Applications Software User interface User friendly Dispensing Protocol optimization flexible parameters New protocol development Characteristics ChIP seq MeDIP seq MethylCap seq hMeDIP IPure Sample preparation Re ChIP MagBisulfite RNA IP Library preparation for NGS platforms Protocols Sample prep MeDIP hMeDIP AS MethylCap ChIP Re ChIP PA Le a 7 i ES IPure MagBisulfite RNA IP Library prep a no diageno e Intuitive touch screen panel Software training not required Automated dispension of assay reagents Antibody coating temperature time mixing speed Immunoprecipitation temperature time mixing speed Washes temperature time mixing speed Achievable by Diagenode product specialist 750W x 740 D x 610H 100 kg 8 Nozzles X Y 7 axis 4 95 C ChIP seq MeDIP seq MethylCap seq hMeDIP IPure Sample preparation Re ChIP MagBisulfite RNA IP PC Software Software training before use Manual dispension of assay reagents Antibody coating temperature time Immunoprecipitation temperature time Achievable by customer after training 1070W x 650 D x 780 H 130 kg 8 Nozzles X Y 7 axis 1429590 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail infogdiagenode com Improved reproducibility DIAGENODE AU
31. ted 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 gPCR 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 America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 30 DIAGENODE AUTO ChIP KIT USER MANUAL Results A Auto ChIP B Auto ChIP H3K9me3 ve locus SAT2 H3K9me3 blue ve locus c fos promoter 40 40 25 25 30 30 nu E E B 15 R 15 10 10 5 5 2 8 1 1 1 1 0 3 0 0 No b IgG H3K9me3 NoAb lg H3K8me3 G Figure AutoChIP ChIP assays was performed using the SX 8G IP Star automated system Diagenode antibodies directed against against H3K9me3 cat pAb 056 050 as well as optimized qPCR prime
32. ts 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 CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO 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 Ct x96input 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 log2 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 calcula
33. uct 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 CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE AUTO ChIP KIT USER MANUAL PAGE 9 Kit Materials Kit Content Two Auto ChIP Kit formats are available and the kit content is sufficient to perform either 16 or 100 Chromatin Immunoprecipitations by using the SX 8G IP Star Automated System The kit content is described in Table 1 Upon receipt store the components at the temperatures indicated in Table Table 1 Kit content Quantity x16 Description Quantity x100 Storage Protein A coated magnetic beads or 220 pl 1500 pl 4 C do not freeze Protein G coated magnetic beads Buffer A 25ml 175 ml 4 C Rabbit IgG or Mouse IgG 15 pl 110 ul 4 1 25 M Glycine 2 ml 15 ml 4 C Buffer B 3 ml 22 ml 4 C Protease inhibitor Mix P 200x 100 ul 700 pl 20 C Buffer C 4 ml 30 ml 4 C DNA Isolation Buffer DIB 4 ml 30 ml 4 C Proteinase K 40 pl 220 pl 20 C Table 2 Reagents available separately Description Reference Description Quantity Storage 1 M Sodium butyrate kch 817 001 1 ml 20 C Protein A coated magnetic beads kch 802 220 The beads are supplied for 16

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