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1. Sheared chromatin Abgang mix Etution butfertinput Ail Immunaprecipitation mix Innovating Epigenetic Solutions H Washes Elution buffer IP Tube Description 200 ul protocol 1 Elution Buffer 1E1 Elution Buffer iE2 94 ul 4 ul 2 Empty 6 Magnetic beads 10 20 ul 4 1x ChIP Buffer iC 1b BSA 5 1 50 100 ul 5 1x ChIP Buffer iC1b BSA 5 1 50 100 ul 6 Ab coating mix 100 ul 7 Immunprecipitation mix 200 ul 8 Wash Buffer iW1 150 ul 9 Wash Buffer iW2 150 ul 10 Wash Buffer iW3 150 ul 11 Wash Buffer iW4 150 ul 12 Elution Buffer 1E1 Elution Buffer 1E2 96 ul 4 ul PAGE 23 This Auto iDeal ChIP seg kit for Transcription Factors 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 NOTE If required NaBu HDAC inhibitior 20mM final concentration or other inhibitors can also be added tothe mmunoprecipitation MIX Running protocol i 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 protocol2. www diagenode com diagendtie PAGE 18 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL 29 Recover the samples in the well 12 and inputs in well 1 Press OK and then YES to start a new run Samples are now ready for purification Finish NOONE e Open the door e Recover the samples and remove the consumables Do you want to start again a protocol Leeeeeeee Sample 11 SD input If you set diageno diageng e Innovating Epigenetic Solutions PAGE 19 STEP 4 Elution decross linking and DNA isolation After the reverse crosslinking DNA purification is performed using our simplified and validated Auto IPure reagents included in the Auto iDeal ChIP seq kit for Transcription Factors and the related protocol on the P Star 30 Select Protocols icon and then IPure category 31 Select IPure protocol for an elution in 50 ul and I Pure seq protocol for an elution in 25 ul NOTE If you plan to run between 1 and 8 samples chose IPure_08 or IPure seq_08 If you plan to run between 9 and 16 samples chose IPure_16 or IPure seq_ 16 If you plan to run between 17 and 24 samples chose IPure_24 or IPure seq_24 32 Setup the exact number of samples for your experiment Each IP and input has to be counted as a sample NOTE The Peltier Block is now cooling down to 4 C to keep your samples cold 33 Setup all the plastics on the platform according to the screen
3. Re suspended the pellet in 20 ul of TE buffer Run samples 20 ul of DNA 4 ul of 6x loading dye ina 1 5 agarose gel Innovating Epigenetic Solutions 5 wo 13 mw H 40 50 edd i000 2000 10980 bp U 1000 2000 2000 A Cc Figure 6 Superior chromatin shearing results with the Bioruptor Plus using buffers and protocol of the Diagenode iDeal ChIP seg kit Hela cells were fixed with 1 formaldehyde for 8 min at RT Nucleus isolation of five million fresh or frozen stored at 80 C cells are performed using buffers of the Diagenode iDeal ChIP seq kit Cat No CO1010050 and are then resuspended in 200ul of Shearing Buffer iS1 prior to chromatin shearing Samples are sheared during 1 2 or 3 rounds of 10 cycles of 30 sec ON 30 sec OFF with the Bioruptor Plus combined with the Bioruptor Water cooler at HIGH power setting position H For optimal results samples are vortexed before and after performing 10 sonication cycles followed by a short centrifugation at 4 C All samples were treated with RNase see Additional Protocols Panel A 10 ul of DNA equivalent to 300 ng are analyzed on a 1 5 agarose gel Panel B and C Sample 3 3x 10 min was analyzed on Bioanalyzer 2100 using DNA High Sensitivity chip The default log scaled Bioanalyzer output can be seen in Panel A while Panel C represents their linear transformation for better vizualisation Out of range fragments were not shown in this gra
4. Table 3 Protocol Name ChIP IPure 200 protocol Reagent Preparation Th Magnetic Bead Washes 30 min Ab coating 3 hours IP reaction 10 15 hours Washes and elution Th Add reagents 15 min DNA isolation or reverse cross linking 4h reverse cross linking DNA recovery ds DNA Input required is sheared chromatin ready to ChlIP Switch on the computer ee SE Start SX 8G V32 software through the following icon gt Place the prepared tube strip on the right cooling heating block of the workstation Switch on the SX 8G IP Star The power switch is on the right side of the instrument www diagenode com diagendtie PAGE 24 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL ETETETT LETETTE ee eeeree ee 5 Close the workstation door and lock it using the following icon gt ns 6 Press the following icon gt ire Select ChIP IPure 8 200 protocol X8G V52 Ver0 7 ChIP_Small_Demo HLD x IMPORTANT NOTE he program ended successfully If the ChIP protocols do not appear in the screen diagendtie 1 Open the SX 8V52 directory P Si ai 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 In re
5. Ethanol e 70 Ethanol e DNA precipitant Cat No C03030002 e DNA co precipitant Cat No C03030001 Take an aliquot of 50 ul of sheared chromatin and spin the chromatin at 12 000 rpm for 10 min at 4 C Transfer the Supernatant to a new tube for chromatin analysis Prepare RNase cocktail dilution e g Ambion AM 2286 A dilute 1ul of cocktail in 150 ul of ChIP seg grade water Add 2 ul of diluted RNase cocktail Incubate 1h at 37 C Add 50 ul of elution buffer iE1 Add 4 ul of elution buffer 1E2 mix thoroughly Incubate samples at 65 C for 4h or overnight Extract DNA once with an equal volume of phenol chloroform isoamyl alcohol 25 24 1 Incubate the sample at RT for 10 min on a rotating wheel Centrifuge for 2 min at 13 000 rpm at RT Transfer the top aqueous phase into a new 1 9 ml tube Add 1 volume of chloroform isoamyl alcohol 24 1 Incubate the sample at RT for 10 min on a rotating wheel Centrifuge for 2 min at 13 000 rpm at RT Transfer the top aqueous phase into a new 1 9 ml tube Precipitate the DNA by adding 10 ul DNA precipitant 5 ul of co precipitant and 500 ul of cold 100 ethanol to the sample Incubate at 80 C for 30 min Centrifuge for 25 min at 13 000rpm at 4 C Carefully remove the supernatant and add 500 ul of ice cold 70 ethanol to the pellet Centrifuge for 10 min at 13 000 rpm at 4 C Carefully remove the supernatant leave tubes open for 30 min at RT to evaporate the remaining ethanol
6. For the positive control antibody CTCF the recovery of the positive control target H19 imprinting control region is expected to be approximately 5 although this will depend on the cell type used The recovery of the negative control target Myoglobin exon 2 locus should be below 0 5 6 Figure 4 ChIP was performed on human HeLa cells m H19 imprinting using the control antibodies from the iDeal ChIP seq 5 control PAON kit for Transcription Factors Sheared chromatin from m Myoglobin 4 million cells 0 5 ul of the positive control antibody Bron and 1 ul of the negative IgG control were used per 4 IP Quantitative PCR was performed with the positive control H19 imprinting control region and the negative control Myoglobin exon 2 primer sets from the kit The recovery expressed as a of input the relative amount of immunoprecipitated DNA compared to input DNA after gPCR analysis of input oO CTCF IgG How to perform Automated ChIP on the IP Star Compact IP STAR COMPACT Protocol STEP 1 Cell collection and DNA protein cross linking 1 Dilute formaldehyde in Fixation buffer to a final concentration of 11 e g add 5 ml of a 37 formaldehyde solution to 11 8 ml Fixation buffer For a 1175 culture flask you will need 2 ml of diluted formaldehyde NOTE When studying inducible transcription factors or cofactors it is recommended to perform
7. settings of the sonicator apparatus l Critical points for shearing 1 Not to start with a too high concentration of cells 15 x 10e6 cells ml or less is ok Chromatin optimization 2 Keep samples cold 4 C shearing Shear the samples of chromatin using the Bioruptor Pico from Diagenode Maintain temperature of the samples close to 0 C The chromatin shearing needs to be optimized for each cell type A troubleshooting guide for Bioruptor chromatin shearing is available at Diagenode Innovating Epigenetic Solutions Purify the DNA from the sheared chromatin as described in the kit protocol to analyse the shearing Extract total DNA from an aliquot of sheared chromatin and run on 1 agarose gel stain with EtBr In order to analyse the sheared chromatin on gel take DNA purified from the sheared chromatin input prepared at STEP 3 Some unsheared chromatin can be analysed on gel as well purify it as done with the input sample see 6 Additional protocols section Chromatin eqvivalent to 100 000 cells one million cells or more can for sure be visualized on a gel Sheared chromatin Loading of large quantities of DNA on agarose gel can lead to poor quality pictures analysis Do not load too much DNA ona gel which do not reflect the real DNA fragmentation The DNA amount to load depends on the size of the well and on the gel size Do not use more than 1 1 5 agarose gel and run slowly Volt cm and
8. we recommend analysing the IP d DNA by qPCR using at least 1 positive and 1 negative control target The kit contains a positive and negative control primer pair which can be used for the CICF positive control antibody in SYBR Green gPCR assay using the protocol described in the manual Use your own method of choice for analysing the appropriate control targets for your antibodies of interest In order to have sufficient DNA left for sequencing we recommend not using more than 10 of the total IP d DNA for qPCR You can dilute the DNA 1 10 or more to perform sufficient PCR reactions PCR reactions should be performed at least in duplicate although performing them in triplicate is recommended to be able to Identify potential outliers 8 Quantitative PCR data interpretation The efficiency of chromatin immunoprecipitation of particular genomic loci can be expressed as the recovery of that locus calculated as the percentage of the input the relative amount of immunoprecipitated DNA compared to input DNA If the amount used for the input was 1 of the amount used for ChIP the recovery can be calculated as follows recovery 2 Ctinput Ctsample Lane and ae are the threshold cycles from the exponential phase of the qPCR for the IP d DNA sample and Input respectively This equation assumes that the PCR is 100 efficient amplification efficiency 2 For accurate results the real amplification efficiency If known should be used
9. 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 Formaldehyde 37 Molecular Grade e Phosphate buffered saline PBS buffer e 1 M Sodium butyrate NaBu Cat No C12020010 optional e 100 isopropanol e RNase DNase free 1 5 ml tubes e qPCR SYBR Green Mastermix e Reagents for library preparation cluster generation Illumina or ePCR lon TorrentTM PGMTM and sequencing e Quant IT dsDNA HS assay kit Invitrogen e Diagenode ChIP cross link Gold Cat N C01019027 Optional For library preparation we highly recommend e High Resolution Library Preparation kit Cat No C05010023 e MicroPlex Library Preparation kit v2 Cat No C05010012 12 reactions 12 indices Cat No C05010013 48 reactions 12 indices Equipment e Diagenode DiaMag02 magnetic rack Cat No B04000001 e Diagenode Bioruptor sonication device Cat No Standard B01010001 Plus B01020001 and Pico B01060001 e Diagenode 1 5 ml TPX Microtubes optimized for chromatin shearing with Bioruptor Standard or Plus Cat No C30010010 or 1 5 ml Bioruptor Microtubes with Caps Cat No C30010016 optimized for chromatin shearing with Bioruptor Pico e Refrigerated centrifuge for 1 5 ml 19 ml and 50 ml tubes e Cell counter e Vortex e Thermomixer e Qubit system Invitrogen e qPCR cycler e For tissues o Petri dishes PAGE 9
10. 6 Magnet OFF 0 1 9 7 Interium Height Z Move PLGO C Documents and SettingsWgniacioiMy Completed 1 10 1 Right blacktvell5 A 1 gt C Documents and Settingsilgniacio My WakeUp 1 10 2 Action Z B na 1 10 3 Pre asp C 1 10 4 Beads resuspend Des 5 Pre Dsp E Eten 2 Volume Mixing Clas 1 11 3 Magnet ON H 1 11 4 Volume Mixing 1 11 45 Air disp J 1 11 6 Magnet OFF 0 Kee 1 11 7 Air Dsp hoes 1 11 8 Interium Height Z Move Repeat f vait msec Stack DUP Liq_in POP P_SPEED_H Asp Speed Wait_msec 200 waittime msec Stack DUP 1 12 1 0 1 12 2 IP reaction 0 1 1 3 0 1 13 1 Right block vVell6 1 13 2 Action Z 1 13 3 Beads resuspend Liq_out POP P_SPEED_H Disp Speed 1 13 4 IP reaction 0 Pass_time 1 13 45 Air Dsp IF_Goto LE 7 200 Mixing Time Sec Repeat 1 13 68 Interium Height Z Move Stack Drop 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 8 Reverse crosslinking After the IP washes the following window will be appear Attention Please x P sss CAUTION gt gt gt gt Open the door Add tul input in well 1 And put the cap on the PCR tube OK 1 Add 1 Input to well 1 2 ul of sheared chromatin 2 Close the tube strip with the corresponding caps 3 Press OK
11. C DiaMag protein A coated magnetic beads 800 ul 3 3 ml 4 C DO NOT FREEZE Wash buffer iW1 10 ml 42 ml 4 C Wash buffer iw2 10 ml 42 ml 4 C Wash buffer iW3 10 ml 42 ml 4 C Wash buffer iW4 10 ml 42 ml 4 C ChIP seq grade water 15 ml 120 ml 4 C Elution Buffer 1E2 940 ul 720 ul 4 C Fixation buffer 6 1 ml 26 ml 4 C Wash buffer 1 w o iso propanol 2ml 8 ml 4 C Wash buffer 2 w o iso propanol 2ml 8 ml 4 C Buffer C 1 7 ml 8 ml 4 C IPure Beads v2 260 ul 1 6 ml 4 C DO NOT FREEZE Elution Buffer iE1 12 ml 16 ml 4 C 5x ChIP Buffer iC1b 4 4 ml 32 ml 4 C Lysis Buffer iL1b 110 ml 470 ml 4 C Lysis Buffer iL2 66 ml 280 ml 4 C Table 2 Kits and Modules available separately Description Reference Quantity ChIP Cross ling Gold C01019027 600 ul Chromatin Shearing Optimization Kit Low SDS for TFs C01020013 20 6 XMS Auto Pure kit v2 x100 C03010010 100 rnx Innovating Epigenetic Solutions Table 3 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 microtube for SX 8G IP Star Compact C30010014 100 Large reagent container for SX 8G IP Star Compact C30020004
12. The information in this guide is subject to change without notice Diagenode and or its affiliates reserve the right to change products and services at any time to incorporate the latest technological develop ments Although this guide has been prepared with every precaution to ensure accuracy Diagenode and or its affiliates assume no liability for any errors or omissions nor for any damages resulting from the application or use of this informa tion Diagenode welcomes customer input on corrections and suggestions for improvement NOTICE TO PURCHASER LIMITED LICENSE The information provided herein is owned by Diagenode and or its affiliates Subject to the terms and conditions that govern your use of such products and information Diagenode and or its affiliates grant you a nonexclusive nontransfer able non sublicensable license to use such products and information only in accordance with the manuals and written instructions provided by Diagenode and or its affiliates You understand and agree that except as expressly set forth in the terms and conditions governing your use of such products that no right or license to any patent or other intellectual property owned or licensable by Diagenode and or its affiliates is conveyed or implied by providing these products In particular no right or license is conveyed or implied to use these products in combination with any product not provided or licensed to you by Diagenode and or its affiliates for such
13. avadis ngs com e Geneious http www geneious com web genelous genelous pro e GenoMiner http www astridbio com genominer html e GenoMatix http www genomatix de G oe pe eel oe Be ee eee ee ee ee ee ee ee M ee Mean GO corded Eh TH p mpe oe ie n m ee e a ar reer ie a a IPS in VAE D BOA GAPDH COPETA LARCH Ci REPI ADH Figure 5 Various stages of bioinformatics data analysis Representative images made during bioinformatics analysis of ChlP seq data A The reads are accumulating around the binding site to form a peak like structure in the coverage graph Peak callers are used to detect these peaks B A quality control software like FastQC anlyses numerous parameters that can help us assess the goodness of sequencing Here we can monitor the GC content distribution C Descriptive statistics and annotation output by GREAT D Transcription factors tend to produce sharp peaks upper red band while broad enrichments are characteristic of many histone modifications lower green band www diagenode com diagendue PAGE 30 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Aditional protocols Sheared chromatin analysis Reagents not supplied with the iDeal ChIP seq kit Se er ef 10 11 12 13 14 15 16 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
14. buffer iS1b Prepare 1 ml of complete shearing buffer per tube of 15 million cells Keep on ice 13 Add 1 ml of complete Shearing buffer iS1b to 15 Million cells Resuspend the cells by pipetting up and down several times The final cell concentration should be 1 5 Million cells per 100 ul buffer iS1b Split into aliquots of 100 to 300 PAGE 21 www diagenode com diagendtie PAGE 22 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL ul and transfer the cell suspension to 1 5 ml TPX microtubes Diagenode cat No M 50001 when using the Bioruptor Standard or Plus or to 1 5 ml Bioruptor Microtubes with Caps Cat No C30010016 optimized for chromatin shearing with the Bioruptor Pico 14 Shear the chromatin by sonication using the Bioruptor When using the Bioruptor Standard or Plus shear for 1 to 3 runs of 10 cycles 30 seconds ON 30 seconds OFF each at nigh power setting Briefly vortex and spin between STEP 3 Magnetic immunoprecipitation conditions will work excellent for many cell types However depending on the cell type and Bioruptor system used optimisation may be required 15 Centrifuge at 13 000 rpm 16 000 x g for 10 minutes and collect the supernatant which contains the sheared chromatin Use the chromatin immediately in immunoprecipitation or store it at 80 C for up to 2 months If desired the chromatin shearing effciciency can be analysed at this step see the protocol in A
15. 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 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 iDeal ChIP seg kit for Transcription Factors The Auto iDeal ChIP seg kit for Transcription Factors was developed to enhance the utility of the ChIP procedure allowing one to perform many more ChIPs per day and per week The enti
16. the fixation using the ChIP cross link Gold C01019027 in addition to the formaldehyde fixation 2 Add 1 10 volume of the diluted formaldehyde directly to the cell culture medium 3 Incubate the cells for 10 to 20 minutes at room temperature with gentle shaking The fixation time can depend on your target of interest 4 Add 1 10 volume of Glycine to the cell culture medium to stop the fixation Incubate for 5 minutes at room temperature with gentle shaking NOTE The fixed cells can be stored at 80 C for up to 4 months However we strongly recommend using freshly fixed cells for preparation of sheared chromatin prior to ChIP for ChlP sequencing STEP 2 Cell lysis and chromatin shearing For adherent cells 5a Remove the medium and wash the cells once with 20 ml of PBS Keep everything at 4 C from now on 6a Add 5 ml of cold Lysis buffer iL1b to the plate and collect the cells by scraping 7a Add an additional volume of Lysis buffer iL1b to rinse the flask and add this to the collected cells The total volume of Lysis buffer iL1b should be about 10 ml per 107 cells e g for a T175 culture flask 25 milllion cells rinse with an additional 20 ml of buffer iL1b 8a Incubate at 4 C for 20 minutes For suspension cells 5b Pellet the cells by centrifugation at 1 600 rpm and 4 C for 5 minutes Discard the cell culture medium 6b Wash the cells once with PBS Resuspend the cells in 20 ml of PBS centrifuge at 1 600 rom an
17. 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 www diagenode com diagendtie PAGE 8 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Kit materials The Auto iDeal ChIP seq kit for Transcription Factors x100 contains reagents to perform 17 different chromatin preparations 100 Chromatin Immunoprecipitations and DNA Purification by using the IP Star s Automated System The Auto iDeal ChIP seq kit for Transcription Factors x24 contains reagents to perform 4 different chromatin preparations 24 Chromatin Immunoprecipitations and DNA Purification by using the IP Star s Automated System The kit content Is described in Table 1 Upon receipt store the components at the temperatures Indicated in Table 1 Table 1 Kit content Description Quantity x24 Quantity x100 Storage Protease inhibitor cocktail 72 ul 385 ul 20 C 5 BSA DNA free 990 ul 3 5 ml 20 C Rabbit IgG 15 ug 1 ug l 40 ug 1 ug ul 20 C ChIP seq grade CTCF antibody 10 ug 2 5 ug pl 40 ug 2 5 g t 20 C ChIP seq grade H19 imprinting control region primer pair 50 ul 500 ul 20 C ChIP seq grade Myoglobin exon 2 primer pair 50 ul 500 ul 20 C Carrier 67 ul 320 ul 20 C Glycine 97ml 40 ml 4 C Shearing Buffer iS1b 75 ml 31 ml 4
18. 4 Reverse crosslinking will be performed at 69 C for 4 hours or 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 STEP 4 Elution decross linking and DNA isolation After the reverse crosslinking DNA purification is performed using our simplified and validated Auto IPure reagents included in the Auto iDeal ChIP seg kit for Transcription Factors and the related protocol on the P Star To run this protocol on the P Star please follow the instructions from the manual Auto IPure kit v2 C03010010 Innovating Epigenetic Solutions STEP 5 Quantitative PCR analysis Before sequencing the samples we recommend analysis the IP d DNA by qPCR using at least 1 positive and 1 negative control target The kit contains a positive H19 imprinting control region and negative Myoglobin Exon 2 control primer pair which can be used for the positive control antibody provided in the kit CTCF ChIP seg grade analysing the appropriate control targets for your antibodies of interest 42 Prepare the qPCR mix as follow 20 ul reaction volume using the provided control primer pairs e 10 ul of a 2x SUBR Green qPCR master mix e 1 ul of primer mix e 4 ul of water e 5 ul IP d or input DNA 43 Use the following PCR program 3 to 10 min denaturation step at 95 C please check carefully supplier s recommendations a
19. 80 Fax 1 862 209 4681 orders na diagenode com info na diagenode com FOR A COMPLETE LISTING OF DIAGENODE S INTERNATIONAL DISTRIBUTORS VISIT http www diagenode com company distributors php For rest of the world please contact Diagenode sa www diagenode com DIAGENODE HEADQUARTERS 2015 Diagenode Inc All rights reserved The content of this document cannot be reproduced without prior permission of the authors Bioruptor amp IP Star are registered trademarks of Diagenode MA_Auto iDealChIPseqIF 100rxns V1_06_ 15
20. ACS2 e SICER http home gwu edu wpeng Software htm e ZINBA http code google com p zinba We are extensively using MACS 1 4 1 for our experiments While it is a prominent tool for shorter peaks sometimes it has difficulties with broader regions therefore we recommend you to set a wider local peak background and lower the pvalue cutoff if necessary for histone marks In some cases turning off the local lambda calculation provides a better coverage of broad enrichment islands though this can result in more false positive peaks detected Please refer to the MACS manual http liulab dfci harvard edu MACS README html if you are not sure how to tweak the parameters c Having your peaks you can start decrypting the epigenetic code The visual Inspection is a common first step especially if the aim of your experiment was to See If certain genes have certain histone modifications transcription factors attached or you want to check some positive negative control sites for enrichment Choose the appropriate viewer software according to the output format of your peak caller and your preferences Annotation is always very useful since you can identify biological features that are relevant to your peaks or check if you have the peaks at the expected loci like H3K4me3 enrichments in the promoter regions of active genes You can expand the annotation with a gene ontology pathway analysis of the peak associated genes thus discovering how your transcr
21. Buffer iC1b BSA 5 1 50 Mix for 1 IP 300ul per IP is needed ox ChIP Buffer iC1b 60 ul ChIP seq grade Water 234 ul 5 BSA DNA free 6 ul e If lt 8samples prepare 300 ul excess 1 IP excess e If gt 9 samples prepare 1200 ul excess 4 IP excess 2 Preparation of Ab coating mix Well 6 Antibody x ul 1x ChIP Buffer iC1b BSA 5 1 50 100 x l 200x Protease Inhibitor Cocktail 0 5 ul Use 1 ul 1ug ul of the IgG negative control antibody for the negative control IP If a positive control IP is included in the experiment use 0 5 ul 2 5ug ul of the CTCF positive control antibody 3 Preparation Immunoprecipitation mix Well 7 Sheared chromatin 200 ul BSA 5 4 ul 200x Protease Inhibitor Cocktail 1 ul Keep 2 ul of the sheared chromatin aside for the Input 25 Fill Reagent Racks 1 amp 2 with reagent according to the screen instructions and Press Next Innovating Epigenetic Solutions Reagent information A DIB Elution buffer Beads Wash Buffer 1x ChIP buffer iC1b BSA 5 1 50 Elution buffer Elution Buffer iE1 ul IP wash 1 Wash Buffer iW1 B Beads wash buffer IP wash 2 Wash Buffer iW2 ul IP wash 3 Wash Buffer iW3 C F IP wash No 1 4 IP wash 4 Wash Buffer iW4 ul PAGE 17 26 Check the selected parameters close the door and press Run to start Set confirmation Protocol and Sample Protocol Sample number Configur
22. S eaae 13 SEF S Mae C1 EO OP SCNT O eerie EEE EE ES EIE NEE TTE I EEE EATERIES 14 SEFA Elution decross linkmg and DNA SC a OU vesnwensusenceinontyeacarnearicinncominwtrr NN EE EE ETNE EET EEEE 19 SEP OF Quanitaiive FORAa nalyS S etc sccenerntiacaetetatuedabdatnasonsahl tnnitaclasdemdeelstarsabaiieba dan aarin iere iei akata ia 27 How to perform automated ChIP on the IP Star ceo b bebo bt be tb ett eens 20 STEP 1 Cell collection and DNA protein cross UNKING ecrereecscessi ritesi nirsTene ncn 21 SBE 2 Cel Sls ene Cn Ghia Ui Sea ccicenice wieonpisrapnarsdge EE entrar eaaeisa RAEE 21 Ser oe Magne Ue Mi EMO Ol SCHON eean E RE EEEE ET 22 51 EP 4 Elution GEChOSS lINKING and DNA ISOLATION oec iniaiaiai iiki tudes 26 eS Se aaneen eane a a e dE hla piid dia erea daneler iinis 27 CFU SN UN eea n arer aaa a N EE EN ATRE E R TES T E 27 ChIP seq data analysis recommendations aa e bbb bbb bb tt bet ettttret tenet rene 27 Additonal Protocols Fo oe ence eis a A a a a AE dan Banter aee ai 30 Troubleshooting guide EEEE EEEE EEEE EEEE EEEE EEEE EEEE EEEE EEEE EEEE 32 PAGE 3 www diagenode com diagendtie PAGE 4 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Introduction The Diagenode 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
23. ared Efficient fixation of a protein to chromatin in vivo is a crucial step for ChIP The extent of cross linking is probably the most important parameter Two major problems concerning the subsequent immunoprecipitation step should be taken into account 1 an excess of cross linking can result in the loss of material or reduced antigen availability in chromatin or both 2 the relative sensitivity of the antigen epitopes to formaldehyde It is essential to perform the cross linking step with care It is essential to quench the formaldehyde Use glycine to stop the fixation quench formaldehyde with 125 mM glycine for 5 minutes at room temperature add 1 10 volume of 1 25M glycine Alternatively wash the fixed cells properly and make sure you get rid of ALL the formaldehyde Temperature is critical Perform cell lysis at 4 C cold room or on ice Keep the samples ice cold at all times during the cell lysis and use ice cold buffers see STEP 3 Cell lysis Protein degradation during lysis can ae ene Add the protease inhibitors to the lysis buffer immediately before use Cell type Kitorotocotvsiidatior HeLa NCCIT 293T Chondrocytes P19 ASC adipose stem cells and Kerationocytes yP P l have been used to validate the Magnetic ChIP protocol Optimal shearing conditions are Shearing conditions for each cell type must be optimized from cell collection fixation to important for ChIP efficiency shearing method
24. ask and add this to the collected cells The total volume of Lysis buffer iL1b should be about 10 ml per 10 cells e g for a T175 culture flask 25 milllion cells rinse with an additional 20 ml of buffer iL1b 8a Incubate at 4 C for 20 minutes For suspension cells 5b Pellet the cells by centrifugation at 1 600 rpm and 4 C for 5 minutes Discard the cell culture medium 6b Wash the cells once with PBS Resuspend the cells in 20 ml of PBS centrifuge at 1 600 rom and 4 C for 5 minutes and discard the supernatant Keep everything at 4 C from now on 7b Add 1 ml ice cold lysis buffer iL1b to the cell pellet and resuspend the cells by pipetting up and down several times Add an additional amount of buffer iL1b to obtain a total volume of about 10 ml per 10 cells e g for a T175 culture flask 25 milllion cells add an additional 24 ml of buffer iL1b 8b Incubate at 4 C for 20 minutes 9 Pellet the cells by centrifugation at 1 600 rpm for 5 minutes and 4 C and discard the supernatant 10 Add 1 ml ice cold Lysis buffer iL2 to the cell pellet and resuspend the cells by pipetting up and down several times Add an additional amount of buffer iL2 and incubate for 10 minutes at 4 C with gentle mixing For 25 million cells the total amount of iL2 should be 15 ml 11 Pellet the cells again by centrifugation for 5 minutes at 1 600 rpm 500 x g and 4 C and discard supernatant 12 Add 200x protease inhibitor cocktail to Shearing
25. ation Ab coating IP reaction Washes Current temperature Left block Right block Cc diagenctie 27 ChIP is running The Remaining time calculation will give you an estimation of the processing time of your experiment Running status Protocol name ChIP_8 DIB 100 D Washing HI Remaining time 01 11 15 Current temperature value Left block 4 0 C Rightblock 25 0 C diagenctie 28 The next morning after the overnight incubation Recover the sample tubes and place them on the DiaMag02 magnetic rack Cat No B04000001 Keep the supernatant and discard the beads CAUTION e Setup the Input in the 1st well INPUT 2 ul sheared chromatin 94 ul Elution Buffer iE1 e Add 4ulof Elution Buffer iE2 5M NaCl in all the samples well 12 and inputs well 1 e Close the tubes with the caps close the door and press OK e Add input sample in well 1 and ution buffer Brrr 4 ul 5M NaCl in wells 1 and 12 weeeeseeee 200000000 LLLI e After that put caps on tubes 700000000 6 CCCCCe 500000000 s ececeeoe 2300000000 2 WEG 1 diageno e NOTE 1 optionnal Proteinase K can be added for the reverse crosslinking However Diagenode does not provide Proteinase K NOTE 2 optionnal 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 ChIP seq experiments However Diagenode does not provide RNase
26. bout Taq polymerase activation time followed by 40 cycles of 30 seconds at 95 C 30 seconds at 60 C and 30 seconds at 72 C These conditons may require optimization depending on the type of Master Mix or qPCR system used ChiP sequencing The iDeal protocol has been optimized for ChIP seg on an Illumina HiSeq Next Gen sequencer However other sequencing systems such as the Illumina MiSeq or the Life Technologies SOLID systems can also be used Please do not hesitate to contact our customer support team if you have any questions about the design of your ChIP seq experiment or the bioinformatics data analysis Contact for Europe Asia Oceania and Africa ae custsupport diagenode com Contact for North and South America ASK THE custsupport na diagenode com EXPERTS ChIP seg data analysis recommendations To find the captured regions of the genome after sequencing you must perform a a reference alignment followed by b a peak calling then c further data analysis annotation visualization etc to help you find what you are looking for There are abundant software tools for each task that use different approaches to the same problem choose your preferred one considering your dataset and scientific goals The workflows for different sequencers basically differ only in the alignment step since every sequencer has its own characteristic read set short or long fixed or variable length nucleotide or colour code etc a The bu
27. d 4 C for 5 minutes and discard the supernatant Keep everything at 4 C from now on 7b Add 1 mlice cold lysis buffer iL1b to the cell pellet and resuspend the cells by pipetting up and down several times Add an additional amount of buffer iL1b to obtain a total volume of about 10 ml per 10 cells e g for a T175 culture flask 25 milllion cells add an additional 24 ml of buffer iL1b 8b Incubate at 4 C for 20 minutes 9 Pellet the cells by centrifugation at 1 600 rpm for 5 minutes and 4 C and discard the supernatant 10 Add 1 ml ice cold Lysis buffer iL2 to the cell pellet and resuspend the cells by pipetting up and down several times Add an additional amount of buffer iL2 and incubate for 10 minutes at 4 C with gentle mixing For 25 million cells the total amount of iL2 should be 15 ml 11 Pellet the cells again by centrifugation for 5 minutes at 1 600 rpm 500 x g and 4 C and discard supernatant 12 Add 200x protease inhibitor cocktail to Shearing buffer iS1b Prepare 1 ml of complete shearing buffer per tube of 19 million cells Keep on ice 13 Add 1 ml of complete Shearing buffer iS1b to 20 Million cells Resuspend the cells by pipetting up and down several times The final cell concentration should be 2 Million cells per 100 ul buffer iS1b Split into aliquots of 100 to 300 ul and transfer the cell suspension to 1 5 ml TPX microtubes Diagenode cat No M 50001 when using the Bioruptor Standard or Plus or to 1 5 ml Bioru
28. d it is indicated the directory location of the ChIP protocols Before starting the protocol a start confirmation window will appear Press OK and the protocol will run ft Schedule Me ager Yer 0 I Total Action Time 0 Tite CHIP_Small_Demo Active Blocks 1 Innovating Epigenetic Solutions PAGE 25 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 SXBG 5 Ver0 7 ChiP_Smaltl_Demo HLD 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 www diagenode com diagenotie PAGE 26 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL 7 The program will run through the following steps magnetic bead washes IP and IP washes 14 12 14 16 M Simul 1 1 9 2 Volume Mixing During protocol the next window will be displayed indicating the step that the protocol Is processing 1 9 3 Volume Mixing 1 9 4 Volume Mixing Antibody immobilize to beads gt IP reaction gt DNA purification DIB_1 day e 1 9 5 Air Dsp File Name Status 1 9
29. dditional protocols This protocol has been optimised for 4 Million cells per ChIP although it is possible to reduce or increase the amount of cells For using lower amounts of cells simply dilute the chromation in shearing buffer before adding it to the IP reaction For higher cell numbers you can increase the cell concentration in the shearing buffer although this may require an additional optimization of the shearing conditions Therefore we recommend performing separate ChIP s and pool the samples before purification of the DNA 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 1 Prepare 1x ChIP Buffer iC1b BSA 5 1 50 Mix for 1 IP 300ul per IP is needed ox ChIP Buffer iC1b 60 ul ChIP seq grade Water 234 ul 5 BSA DNA free 6 ul e f lt 8samples prepare 300 ul excess 1 IP excess e If gt 9 samples prepare 1200 ul excess 4 IP excess 2 Preparation of Ab coating mix Well 6 Antibody 1x ChIP Buffer iC 1b BSA 5 1 50 x ul 100 x ul 200x Protease Inhibitor Cocktail 0 5 ul Use 1 ul 1ug ul of the IgG negative control antibody for the negative control IP If a positive control IP is included in the experiment use 0 5 ul 2 5ug ul of the CTCF positive control antibody 3 Preparation Immunoprecipitation mix Well 7
30. diageng e Innovating Epigenetic Solutions Auto iDeal ChIP seg kit for Transcription Factor Cat No C01010058 24 rxns Cat No C01010172 100 rxns Version 1106 15 Contacts DIAGENODE HEADQUARTERS 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 orders diagenode com info diagenode com Diagenode Inc USA NORTH AMERICA 400 Morris Avenue Suite 101 Denville NJ 07834 Tel 1 862 209 4680 Fax 1 862 209 4681 orders na diagenode com info na diagenode com For a complete listing of Diagenode s international distributors visit http www diagenode com company distributors php For rest of the world please contact Diagenode sa Diagenode website www diagenode com Content WE OUI C TOs agace cece A eae A E EA oie A E E E E pone acecaseesamearcaniiciasag ES E 4 IP Star and IP Star Compact Systems for automation of epigenetic applications 5 Kit method VT VU rarena r nace sans gt vn ee hae to nun ores aa eaten deen onan Perenea sat T teen den atigess 7 KE OS a A AEEA E A tan otene es 8 Required materials not provided 9 Remarks before starting EEEE EEEE EEEE 10 How to perform automated ChIP on the IP Star Compact aa 12 STEP TGellcollection and DNA protein Cross UNKINGsiccsc cco cesnniaz gui veyrasigds piia nE SBE E aa 13 TEF Z ell yss andi hiromnaun enean eee inean E E a EET EASE AEAT EE EAR IRTEE
31. eared into fragments of 100 to 600 bp Our kits and protocols are optimized for chromatin shearing using the Bioruptor Standard Plus and Pico The maximum volume for shearing with the Bioruptor is 300 ul per 1 5 ml Microtube depending on the specific type We recommend using TPX tubes C30010010 for Bioruptor Standard and Plus as shearing has been shown to be more efficient and reproducible using these tubes For Bioruptor Pico we recommend using 1 5 ml Microtubes with Caps C30010016 The shearing conditions mentioned in the protocol are adequate for a variety of cell types However given that cell types are different we recommend optimizing sonication conditions for each cell type before processing large quantities of cells or samples It is Important to perform an Initial sonication time course experiment to evaluate the extent of chromatin fragmentation A protocol to assess the shearing efficiency can be found in the Additional Protocols section 4 Magnetic beads This kit includes DiaMag Protein A coated magnetic beads Make sure the beads do not dry during the procedure as this will result in reduced performance Keep the beads homogenously in suspension at all times when pipetting Variation in the amount of beads will lead to lower reproducibility Do not freeze the beads The amount of beads needed per IP depends on the amount of antibody used for the IP The protocol below uses 10 20 ul of beads The binding capacity of this a
32. eeeo weeeeeee TIP Rack Left ac Peltier eoeoeece Block eceocecee eceococe 100000000 100000000 eceeeeee eceoecee 3200000000 P a weeeeeoe 200000000 eiai 100000000 190000000 8 7 6 5 HR ma O gD NS om recat Ea w o A A F eee 196 plate 2 4 amp 8 400608060806080 06 22 Fill TIP Rack 1 and 2 if processing 16 samples protocol with tips according to the screen 23 Fill Reagent Racks 1 amp 2 with reagent containers according to the screen 24 Fill Peltier block with your sample antibody and magnetic beads as mentioned her below PAGE 15 www diagenode com diagendtie PAGE 16 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL 10 0000000 00000000 90000000680 8060060060000 500000000 40000000 200000000 This Auto iDeal ChlP seq kit for Transcription Factors 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 PCR tube Well 7 Sample 200 ul Well 6 Ab in buffer 100 wl Well 3 Magnetic beads accordingly Please contact us for advice NOTE If required NaBu HDAC inhibitior 20mM final concentration or other inhibitors can also be added to the chromatin sample Direct ChIP 1 Prepare 1x ChIP
33. ilt in aligners with default settings worked very well for our ChIP seg experiments e g ELAND for Illumina TMAP for PGM If you cannot access them open source tools are also available we have positive experience with BWA http bio bwa sourceforge net If you use a multipurpose aligner do not forget to use settings appropriate to your dataset please consult with the manual of your software b The purpose of the peak calling is to find the enriched regions in the alignment Take extreme care when you choose and set up your peak caller since the outcome can vary widely depending on the used software and its settings We advise you to read the comparative literature and the software manuals to fully understand how a certain program works One of the key features of your data is the expected length of the enrichment regions Transcription factors tend to produce short and sharp peaks while histone marks create broad islands of enrichment A remarkable tool for sharp peak detection is MACS while SICER is dedicated to histone marks and tools like ZINBA can be used PAGE 27 www diagenode com diagendtie PAGE 28 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL for both with decent outcomes MACS 2 is reported to be better suited for histone marks than previous versions The availability of the mentioned softwares e MACS http liulab dfci harvard edu MACS e MACS 2 https github com taoliu MACS tree master M
34. iption factor histone modification is involved in the cell s or the whole organism s life Motif search is almost an obligatory analysis for the sequence specific transcription factors but you may find common motifs among histone modification sites as well so you can check for example if you Indeed have promoter specific motifs in your theoretically promoter specific enrichments A lot of programs including peak callers themselves output descriptive statistics of the peaks measuring for example their enrichment ratios significances width heights reads in peaks This characterization helps you better understand your data which is essential for most applications a typical example is the comparison of performance of different sample preparation protocols or different sequencer setups The final recommended analysis type is the comparative analysis We encourage scientists to use replicates in their experiments removing peaks that are not common could effectively reduce false positives You can also use a validated reference set of peaks for comparisons but that is rarely available Additionally if you have other biologically relevant data from your samples it is wise to compare and Integrate them For example an RNA seqg dataset is a great source of validation for histone marks that are supposed to regulate gene expression Recommended free tools for the peak analysis e IGV visualization http www broadinstitute org igv e UCSC Genome Br
35. layout 34 Add 2 ul of carrier to each IP and input sample and place them on the Left block 35 Resuspend and dispense 10 ul of magnetic beads IPure for each sample on the 96 well plate NOTE Keep the magnetic beads in liquid suspension during storage at 4 C and at all handling steps as drying will result in reduced performance Make sure the beads are homogeneously in suspension at all the time during pipetting steps because the beads are precipitating rapidly 36 Dilute Wash Buffers 1 1 with isopropanol Wash buffer 1 Wash buffer 2 24 rxns 100 rxns 24 rxns 100 rxns Wash buffer 1w o isopropanol 2ml 8 ml Wash buffer 1w o isopropanol 2ml 8 ml lsopropanol 100 2 ml 8 ml Isopropanol 100 2 ml 8 ml Total volume 4 ml 16 ml Total volume 4 ml 16 ml 37 Dispense Wash Buffers 1 amp 2 with Isopropanol in the appropriate container in the P Star 38 Dispense Buffer C in the appropriate container in the IP Star 39 Press Run to start End e Open the door e Recover the samples and remove the consumables Left block 200000000 100000000 10 8 8e0e000 300000000 4 300000000 200000000 1 eeeeee0e diagenc e 40 Atthe end ofthe run recover your samples on the left block at 4 C 41 Press OK remove the consumables and switch off the IP Star www diagenode com diagendtie How to perform Automated ChIP on the IP Star IP STAR 42 Place the DNA o
36. les per year very reliably Customer The IP Star reduces our processing time down from one day of manual work to just one Feedback overnight 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 Innovating Epigenetic Solutions PAGE 5 IP Star and IP Star Compact Systems for automation of epigenetic applications Diagenode has developed two automated platforms IP Star and 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 P Star and 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 IP Star Compact P Star f W 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 amoun
37. mount Is approximately 2 5 5 ug of antibody With most of Diagenode s high quality ChIP seq grade antibodies the recommended amount to use is 1 to 2 ug per IP reaction Therefore you can reduce the amount of beads accordingly 5 Negative and positive IP controls IgG and control Ab The kit contains a negative IgG and a positive CTCF control antibody We recommend including one IgG negative IP control in each series of ChIP reactions We also recommend using the positive control ChIP seg grade CTCF antibody at least once The kit also contains qPCR primer pairs for amplification of a positive and negative control target for CTCF H19 imprinting control region and Myoglobin exon 2 respectively 6 Quantification Determine the concentration of the IP d DNA after the ChIP with a highly sensitive method such as the Quant IT dsDNA HS assay kit on the Qubit system from Invitrogen PicoGreen Is also suitable but UV spectrophotometric methods such as the NanoDrop are usually not sufficiently sensitive In most cases It is sufficient to use approximately 10 of the IP d material for quantification The expected DNA yield will be dependent on different factors such as the cell type the quality of the antibody used and the antibody target The expected DNA yield obtained with the positive control CTCF antibody on Innovating Epigenetic Solutions 4 000 000 HeLa cells is approximately 20 ng 7 Quantitative PCR Before sequencing the samples
38. n ice and proceed to any desired downstream applications or store it at 20 C or 80 C until further use STEP 1 Cell collection and DNA protein cross linking Protocol 1 Dilute formaldehyde in Fixation buffer to a final concentration of 11 e g add 5 ml of a 37 formaldehyde solution to 11 8 ml Fixation buffer For a 1175 culture flask you will need 2 ml of diluted formaldehyde NOTE When studying inducible transcription factors or cofactors it is recommended to perform the fixation using the ChIP cross link Gold in addition to the formaldehyde fixation 2 Add 1 10 volume of the diluted formaldehyde directly to the cell culture medium 3 Incubate the cells for 10 to 20 minutes at room temperature with gentle shaking The fixation time can depend on your target of interest 4 Add 1 10 volume of Glycine to the cell culture medium to stop the fixation Incubate for 5 minutes at room temperature STEP 2a Cell lysis and chromatin shearing with gentle shaking NOTE The fixed cells can be stored at 80 C for up to 4 months However we strongly recommend using freshly fixed cells for preparation of sheared chromatin prior to ChIP for ChlP sequencing For adherent cells 5a Remove the medium and wash the cells once with 20 ml of PBS Keep everything at 4 C from now on 6a Add 5 ml of cold Lysis buffer iL1b to the plate and collect the cells by scraping 7a Add an additional volume of Lysis buffer iL1b to rinse the fl
39. n of the shearing conditions Therefore we recommend performing separate ChIP s and pool the samples before purification of the DNA 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 16 Switch ON the IP Star Compact 17 Select Protocols icon and then ChIP category 18 Select Direct method and then ChIP_IPure_200_D protocol in the list ChIP_8 IPure_200_D A ChIP_16_IPure_200_D NOTE If you plan to run between 1 and 8 samples chose ChIP_IPure_8_200_D If you plan to run between 9 and 16 samples chose ChIP_IPure_16_200_D Innovating Epigenetic Solutions 19 Setup the exact number of samples for your experiment Each IP has to be counted as a sample Input is not a sample NOTE The Peltier Block is now cooling down to 4 C to keep your samples cold 20 Setup the parameters for your ChIP experiment and press Next Setup the Ab coating step to 3 hours Setup the IP reaction step to 10 15 hours overnight Setup the Washes step to 5 10 min Configuration Mixing time Temperature Mix Speed Ab coating IP reaction Washes 21 Setup all the plastics on the platform according to the screen layout KYYYYYYTY KYYYYYYTY eeeoaeoeoeee eeeoeoeeeo ZEKIK 2 eeeee0ee0 eeceoeooe ee eeeoaoa eee neeseseoeoeeo neeeeeeeo eeeoaoae eo wesee
40. o True MicroChIP kit x16 C01010140 16 rxns Auto True MicroChIP amp MicroPlex Library Preparation Package C01010141 16 ChIP rxns amp 12 library prep rxns MicroPlex Library Preparation kit v2 x12 12 indices C05010012 12 rxns MicroPlex Library Preparation kit v2 x48 48 indices C05010014 48 rxns Auto iDeal ChIP seqg kit for Histones x 24 C01010057 i 24 rxns Auto iDeal ChIP seqg kit for Histones x100 C01010171 100 rxns Auto iDeal ChIP seq kit for Transcription Factors x24 C01010058 24 rxns Auto iDeal ChIP seg kit for Transcription Factors x100 C01010172 100 rxns Auto Plant ChIP seq kit x24 C01010151 24 rxns Auto MeDIP kit x16 C02010011 AF Auto01 0016 16 rxns Auto MeDIP kit x100 C02010012 AF Auto01 0100 100 rxns iDeal Library Preparation Kit x24 incl Index Primer Set 1 C05010020 l 24 rxns Auto hMeDIP kit x16 C02010033 AF Auto02 0016 16 rxns Auto MethylCap x48 C02020011 AF Auto01 0048 48 rxns Auto Pure kit v2 x100 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 www diagenode com 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 orders diagenode com info diagenode com DIAGENODE INC USA NORTH AMERICA 400 Morris Avenue Suite 101 Denville NJ 07834 Tel 1 862 209 46
41. or the correct period of time at the right temperature and with the correct formaldehyde concentration e g incubate for 10 20 minutes at room temperature with 1 formaldehyde final concentration weight volume Also use high quality fresh formaldehyde Proteins have unique ways of interacting with the DNA Some proteins are not directly bound to the DNA but interact with other DNA associated proteins Very short or very long cross linking time can lead to DNA loss and or elevated background therefore the optimal cross linking time should be found empirically as maximal specificity and efficiency of ChIP Cross linking Both cross linking time and formaldehyde concentration are critical Cross linking can affect both efficiency of chromatin shearing and efficiency of specific antigen immunoprecipitation Shorter cross linking times 5 to 10 minutes and or lower formaldehyde concentrations lt 1 weight volume may improve shearing efficiency while for some proteins especially those that do not directly bind DNA this might reduce the efficiency of cross linking and thus the yield of precipitated chromatin The optimal duration of cross linking varies between cell type and protein of interest It is possible to optimize the fixation step by testing different incubation times such as 10 20 and 30 minutes Do not cross link for longer than 30 minutes as cross links of more than 30 minutes can not be efficiently she
42. owser visualization http genome ucsc edu e HOMER motif search annotation gene ontology comparison statistics http biowhat ucsd edu homer e PinkThing annotation conservation comparison gene ontology statistics http pinkthing cmbi ru nl e GREAT annotation statistics http great stanford edu When analysing ChIP seq please always keep an eye on sequencing quality and the performance of the software tools used for analysis For example with a low quality sequencing with a lot of read errors you will have a hard time finding the peaks you are looking for despite your excellent IP d DNA To control the quality use the vendor supplied software and metrics like the ones available in the Illumina pipeline for GA Il Open source tools can also be used e g the FastQC by Babraham Institute http www bioinformatics bbsrc ac uk projects fastac Throughout this chapter we recommended some free tools because they are accessible for everyone and we have tested most of them Please note that there are commercial softwares for the same purposes as well most of Innovating Epigenetic Solutions PAGE 29 them capable of performing several tasks or even a complete ChIP seq workflow Here are a few examples that we know of but we have not tested them e CLC Genomics Workbench http clcbio com e Partek Genomics Suite http www partek com partekgs e NextGENe http www softgenetics com NextGENe html e Avadis NGS http www
43. pecific peptide for about 30 minutes at room temperature before use in the IP incubation mix Use the blocked antibody as a negative control in parallel with the unblocked antibody PAGE 33 www diagenode com diagendtie PAGE 34 Innovating Epigenetic Solutions DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL How many negative controls are necessary If multiple antibodies of the same species are to be used with the same chromatin preparation then a single negative ChIP control is sufficient for all of the antibodies used Why is my antibody not working in ChIP Which antibody should use in ChIP Antibody antigen recognition can be significantly affected by the cross linking step resulting in loss of epitope accessibility and or recognition Use ChIP grade antibodies If not available it is recommended to use several antibodies directed against different epitopes of the same protein Verify that the antibodies can work directly in IP on fresh cell extracts Also when testing new antibodies include known ChIP grade antibodies as positive control for your ChIP assay How do choose an antibody for ChIP Be aware of the possible cross reactivity of antibodies Verify by Western blot analysis the antibody specificity Antigen affinity purification can be used to increase titer and specificity of polyclonal antibodies There is a significant difference in affinity of differen
44. ph In this example the optimal shearing condition corresponds to 3 rounds of 10 cycles 30 sec ON 30 sec OFF PAGE 31 www diagenode com diagendtie PAGE 32 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Troubleshooting guide Error Cause Remedy SX 8G P Star cannot be switched on SX 8G P 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 started SX 8G IP Star shows no movement when a protocol is SX 8G P Star is not switched on Check that the SX 8G IP Star is switched on is started SX 8G6 IP Star shows abnormal movement when a protocol Aspirated liquid drips from the disposable tips 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 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 Critical steps Troubles solutions and comments Cross linking is too weak Cross linking is too strong Make sure you perform the fixation step f
45. ptor Microtubes with Caps Cat No C30010016 optimized for chromatin shearing with the Bioruptor Pico 14 Shear the chromatin by sonication using the Bioruptor When using the Bioruptor Standard or Plus shear for 1 to 3 runs of 10 cycles 30 seconds ON 30 seconds OFF each at high power setting Briefly vortex and spin between PAGE 13 www diagenode com diagendtie PAGE 14 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL each run Shear for 8 10 cycles 30 seconds ON 30 seconds OFF when using the Bioruptor Pico These shearing conditions will work excellent for many cell types However depending on the cell type and Bioruptor system used optimisation may be required 15 Centrifuge at 13 000 rpm 16 000 x g for 10 minutes and collect the supernatant which contains the sheared chromatin Use the chromatin immediately in immunoprecipitation or store it at 80 C for up to 2 months If desired the chromatin shearing effciciency can be analysed at this step see the protocol in Additional protocols This protocol has been optimised for 4 Million cells per ChIP although it is possible to reduce or increase the amount of cells For using lower amounts of cells simply dilute the chromation in shearing buffer before adding it to the IP reaction For higher cell numbers you can Increase the cell concentration in the shearing buffer although this may require an additional optimizatio
46. re 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 iDeal ChIP seg kit for Transcription Factors 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 reaction including antibodies and buffers and also provides you with fewer buffers in comparison with other kits The Auto iDeal ChIP seq kit for Transcription Factors has been validated to perform ChIP seg experiments using antibodies directed against Transcription Factors proteins 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 iDeal ChlP seq kit for Transcription Factors 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 ChIP seq samp
47. t types of immunoglobulins to protein A or G Thererfore in function of the antibody used for your ChIP it is recommended to choose either protein A or protein G coated beads Species immunglobulli isotype ProteinA Protein G IgG1 IgG2 E IgG3 z Antibody in IP Human IgG4 fee IgGM Use anti human IgM IgGF IgGA x 4 Ig61 IgG2a caer Mouse IgG2b 4 Are my antibodies going to bind the IgG3 P protein A or protein G IgGM Use anti mouse IgM IgG1 4 IgG2a 5 Rat IgG2b IgG2c Chicken All isotypes ae Cow All isotypes Goat All isotypes Guinea pig All isotypes Hamster All isotypes Horse All isotypes Pig All isotypes Rabbit All isotypes 4 Sheep All isotypes 4 Freezing Avoid multiple freeze thawing Snap freeze and thaw on ice le g fixed cell pellets and sheared chromatin FOR RESEARCH USE ONLY Not intended for any animal or human therapeutic or diagnostic use 2015 Diagenode SA All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means elec tronic mechanical magnetic optical chemical manual or otherwise without prior written permission from Diagenode SA hereinafter Diagenode
48. time depend on Agarose concentration the gel size Running buffer concentration 1x TAE or TBE is preferred to 0 5x TAE which can lead to smears on agarose gel Sheared Most of the sheared chromatin is to be used in the ChIP experiment but remember How much sheared chromatin do S l chromatin A E RRE that some of the sheared chromatin is needed as control as it corresponds to the input amounts PEREA sample for the ChIP experiment and it can also be checked on agarose gel Antibody binding beads Beads are in suspension Bead storage The provided beads are coated with protein A Resuspend into a uniform suspension before each use Store at 4 C Do not freeze Antibody binding capacity 10 ug 30 pl Protease inhibitors Storage Some inhibitors are unstable in solution The provided P I mix should be kept frozen at 20 C and thawed before use Negative ChIP control s Use non immune IgG in the IP incubation mix Use the non immune IgG fraction from the same species the antibodies were produced in Do not add antibody to the IP Incubation with beads which were not coated with antibodies antibodies could also be used as a negative ChIP control Use a specifically blocked antibody in parallel Use one antibody in ChIP and the same antibody that is blocked with specific peptide To specifically block an antibody pre incubate the antibody with saturating amounts of its epitope s
49. ts Compatible with Diagenode Kits Yy VV VY Vv Vv y4 Reduces cross contamination www diagenode com diagendtie PAGE 6 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Improved reproducibility Our 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 and sheared chromatin were identical for ManChIP and AutoChIP The 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 IP Star is designed to improve the accuracy and the reproducibility of any immunoprecipitiation experiment Man ChIP SDIIgG 0 94 SD IgG 0 17 SD H3K9me3 11 36 ee 1 12 100 0 A 80 0 SD IgG 1 4 SD C Ch 98 62 95 26 SD IgG 0 69 SD H3K9me3 23 84 H3K9me3 2 38 SD IgG 0 09 SD H3K9me3 0 65 D 3 ChIP 1 ChIP 2 ChIP 1 ChIP 2 ChIP 1 ChIP 2 57 83 56 25 60 0 50 70 xe 43 83 44 75 40 0 i 63 20 0 7 1 86 1 62 2 08 12 Auto ChIP SD IgG 0 28 SD H3K9me3 1 6 100 0 90 0 J 80 0 70 0 4 ChIP 1 ChIP 2 ChIP 3 ChIP 4 0 0 4 96 25 54 71 of input IgG H3K9me3 IgG H3K9me3 Innovating Epigenetic Solutions IgG 57 83 H3K9me3 94 34 IgG H3K9me3 Figure 1 Manual ChIP Four different operators ha
50. use Limited Use Label License Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components Is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact infolddiagenode com TRADEMARKS The trademarks mentioned herein are the property of Diagenode or their respective owners Bioruptor is a registered trademark of Diagenode SA Bioanalyzer is a trademark of Agilent Technologies Inc Agencourt and AMPure are registered trademarks of Beckman Coulter Inc Microcon is a registered trademark of Millipore Inc Illumina is a registered trademark of Illumina Inc lon Torrent and Personal Genome Machine are trademarks of Life Technologies Corporation Qubit is a registered trademark of Life Technologies Corporation Ordering information diageng e Innovating Epigenetic Solutions Products Cat No new Cat No old Format IP Star Compact B03000002 UH 002 0001 1 unit Aut
51. ve each performed two ChIP experiments using H3K9mes3 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 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 IP Star are displayed PAGE 7 Kit method overview 2 CHROALATIAVT AA SHEARING Bioruptor Sonication Q CHROMATINAINA Preparation Chromatin Shearing Medium SOS and High SOS CHROMATIN STUDY 8 amp Auto Trua hicroChIP kt Auto Deal ChiP seq kit for Histones Auto iDeal ChiP seq kit for Transcription Factors gt Auto Plant ChiP seq kit DNA METHYLATION LGRARY PREPARATION Auto Pte DAP kit CNA PURIACATION MicroPlex Library Preparation kit Auto hMaDIP kt Auto Pure kit v All py Sngl gt Auto MethyiCan kit magnetic purification iDaal Library Preparation kit bng Ipg 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
52. www diagenode com diagendtie PAGE 10 DIAGENODE AUTO IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Remarks before starting 1 Cell number for cultured cells This protocol has been optimized for ChIP on 4 000 000 cells in 200 ul ChIP reaction For using lower amounts of cells simply dilute the chromation in shearing buffer before adding it to the IP reaction For higher cell numbers you can in crease the cell concentration in the shearing buffer although this may require an additional optimization of the shear ing conditions Therefore we recommend performing separate ChIP s and pool the IP d DNA before purification 2 Cell fixation Formaldehyde is the most commonly used cross linking reagent However formaldehyde is usually not effective to cross link proteins that are not directly bound to the DNA For example chromatin interactions with inducible tran scription factors or with cofactors that interact with DNA through protein protein interactions are not well preserve with formaldehyde When studying this kind of factors we recommend the use of the Diagenode ChIP cross link Gold Cat N C01019027 This reagent is to use in combination with formaldehyde The protocol involves a sequential fixation A first protein protein fixation by the ChIP cross link Gold followed by protein DNA fixation by formaldehyde 3 Shearing optimization and sheared chromatin analysis Before starting the ChIP the chromatin should be sh
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