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1. Description Cat No NEW Cat No OLD Format SX 8G IP Star Compact B03000002 UH 002 0001 1 unit Auto True MicroChlP kit C01010140 16 rxns Auto True MicroChIP amp MicroPlex Library Prep Package CO1010141 Poneman Te aia 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 seq 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 G016 16 rxns Auto Transcription ChIP kit protein G x100 C01010033 AB Auto03 G100 100 rxns Auto ChIP kit protein A x100 C01010011 AB Auto01 A100 100 rxns Auto ChIP kit protein G x100 C01010013 AB Auto01 G100 100 rxns Auto MeDIP kit x16 C02010011 AF Auto01 0016 16 rxns Auto MeDIP kit x100 C02010012 AF Auto01 0100 100 rxns Auto hMeDIP kit x16 C02010033 AF Auto02 0016 16 rxns Auto MethylCap x48 C02020011 AF Auto01 0048 48 rxns Auto 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 Department2. 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 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 info na diagenode com info diagenode com 2013 Diagenode Inc All rights reserved The content of this document cannot be reproduced without prior permission of the authors Bioruptor and IP Star are registered trademarks of Diagenode
3. 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 LL S A Next Gen Sequencing GAPDH Bioruptor Pico oY ENRICHED Magnetic IP Size Selection _ Library Preparation m lumina TruSeq ChIP miPurekit 0 m NEBNext ChIP seq magnetic purification m DNA Isolation Buffer re L Auto MethylCap Kit a Dm STEP 4 8 l DNA Purification m MicroPlex Library Preparation kit 50 pg multiplex manual Figure 3 Diagenode provides a full suite of automated solutions for ChIP experiments For Step 1 we offer products to isolate nuclei and chromatin Step 2 describes reproducible sample shearing with the Bioruptor product line In Step 3 and Step 4 the Diagenode IP Star Compact provides error free walk away automation for all your immunoprecipitation and antibody capture needs Innovating Epigenetic Solutions Kit Materials Kit Content The Auto True MicroChIP kit contains reagents to perform 16 Chromatin Immunoprecipitations by using the SX 8G IP Star and SX 8G IP Star Compact 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 Glycine 5 ml 4 C Lysis Bu
4. 100 ul of sheared chromatin correspond to 10 000 cells then perform 6 reactions in parallel and pool the DNA pellets obtained at Step 14 during respuspension in TE Prepare RNase cocktail dilution e g Ambion AM 2286 A dilute 1 ul of cocktail in 150 ul of water Add 2 ul of diluted RNase cocktail to the chromatin Incubate 1h at 37 C Prepare the Complete Elution Buffer by mixing thoroughly Buffer D E and F Elution module as follow Reagents Volume Buffer D 96 ul Buffer 10 ul Buffer F 4 ul Total volume 110 pl Add 100 ul of the Complete Elution Buffer to each chromatin sample Mix thoroughly and incubate samples at 65 C for 4 hours or overnight Extract DNA once with an equal volume of phenol chloroform isoamyl alcohol 25 24 1 Incubate the samples at RT for 10 minutes on a rotating wheel Centrifuge for 2 minutes at 14 000 xg 13 000 rpm at RT Transfer the top aqueous phase into a new 1 5 ml tube 10 Add 1 volume of chloroform isoamyl alcohol 24 1 Incubate the samples at RT for 10 minutes on a rotating wheel 11 Centrifuge for 2 minutes at 14 000 x g 13 000 rpm at RT Transfer the top aqueous phase into a new 1 5 ml tube 12 Precipitate the DNA by adding 20 ul of meDNA precipitant 5 ul of meDNA co precipitant and 0 5 ml 100 cold ethanol to the sample Incubate at 80 C for 30 minutes Innovating Epigenetic Solutions PAGE 35 13 Centrifuge for 25 minutes at 14 000 x g 13 000 rpm at 4 C Carefu
5. Cell type Number of cells required Chromatin shearing Sonication tips Innovating Epigenetic Solutions Optimize crosslinking time Assure proper fixation time with formaldehyde Optimize formaldehyde concentration Make sure cells disrupt completely Maintain cold temperature during lysis Prevent protein degradation Determine which cell types have previously been validated with the kit Determine number of cells for ChIP Maintain 4 C temperature during shearing Maintain OBC temperature during sonication Optimize SDS concentration Determine amount of sheared chromatin needed for ChIP Dilute the sheared chromatin in ChIP buffer for Immunoselection incubation Determine cell number Sonication conditions with the Bioruptor tips Chromatin shearing with a probe sonicator tips Chromatin shearing with Diagenode modules tips Shearing with other protocols tips Poor crosslinking causes DNA loss elevated background and or reduced antigen availability in chromatin Emperically determine optimal crosslinking time for maximal specificity and efficiency of ChIP The optimal duration of cross linking varies between cell type and protein of interest Short crosslinking time 5 10 minutes may improve shearing efficiency Crosslinking duration should not exceed 30 minutes or shearing will be inefficient Crosslinking may be too weak or too strong without proper fixation time Optimize fixation step e g
6. If the shearing buffer contains 0 75 SDS the sheared chromatin is diluted 3 5 to 4 0 fold in the ChIP buffer Most of the sheared chromatin will be used for ChIP and the input control A small amount will be checked on agarose gel The sheared chromatin is diluted in complete ChIP Buffer tC1 prior to the immunoselection incubation see Step 3 Dilute the sheared chromatin 2 fold Start with 1x10e4 to 1x10e5 cells Shear the samples of chromatin using the Bioruptor for 1 to 5 runs of cycles of 30 seconds ON 30 seconds OFF each These conditions were tested with many mammalian cell lines and were excellent for subsequent ChIP experiments A troubleshooting guide for Bioruptor chromatin shearing is available Probe sonicator Sonicate each sample for 3 x 30 seconds on ice Allow 30 seconds pause on ice between each pulsing session Avoid foaming You can also use the LowCell ChIP kit for shearing 25 ul of complete Buffer B are added per 10 000 cells After 5 minutes lysis on ice 75 ul of HBSS are added and chromatin is sheared in 100 ul aliquots Sheared chromatin have to be diluted 2 times with the complete ChIP Buffer tC1 from the True MicroChlP kit Protocol STEP3 point 21 before adding antibodies When using your own protocol make sure the shearing buffer contains between 0 75 and 1 SDS EDTA 1 10 mM and or EGTA 0 0 5 mM with pH 7 6 8 0 The sheared chromatin is to be diluted in the Complete ChIP Buffer tC1 p
7. 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 transcription factor histone modification is involved in the cell s or the whole organisms 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 Innov
8. e Cell counter e Eppendorf Low retention 1 5 ml Tubes VWR 525 0130 e Bioruptor sonication apparatus e Diagenode 1 5 ml TPX microtubes optimized for chromatin shearing with Bioruptor Cat No M 50050 M 50001 e DiaMage Rotator rotating wheel Cat No VL 100 0001 e Thermomixer 65 C e Vortex e Qubit system e qPCR cycler Innovating Epigenetic Solutions Remarks before starting 1 Cell number and sample manipulation This protocol has been optimized for shearing of 10 000 cells in 100 ul using the Diagenode s Bioruptor and then subsequent IMmunoprecipitation on 10 000 cells in 200 ul Determine the number of IP you will perform and start with fixation of a unique batch of chromatin For example if you would like to perform 4 ChIP on the same chromatin start with fixation of 40 000 cells Add also an extra chromatin preparation to use for the input Due to the low amount of starting material it is critical to avoid sample loss throughout the experiment to ensure reproducible and consistent results Avoid pipetting up and down when adding buffers to samples It is also recommended to use low retention Eppendorf tubes at each step of the protocol to minimize sample loss The use of an automated cell counter is also recommended to reduce variations in the amount of the starting cell number The True MicroChIP kit is also compatible with higher cell numbers Efficient shearing has been validated with the True MicroChIP kit in a
9. incubate for 8 minutes at room temperature with high quality fresh 1 formaldehyde final concentration weight volume Lower formaldehyde concentrations 1 weight volume may improve shearing efficiency For some proteins however especially those that do not directly bind DNA this might reduce crosslinking efficiency and thus the yield of precipitated chromatin Empirically determine the formaldehyde concentration as some antigen epitopes may be more Sensitive to formaldehyde Do not use too many cells per amount of lysis buffer w v so that cells can be completely disrupted Follow the instructions in the protocol Perform cell lysis at 4 C cold room or on ice Always keep the samples ice cold during cell lysis and use cold buffers Add the protease inhibitors to the lysis buffer immediately before use HeLa have been used to validate this magnetic ChIP protocol The number of cells for ChIP is determined by cell type protein of interest and antibodies used Use chromatin from 10 000 cells per ChIP In some cases chromatin from up to 100 000 cells may be needed You may need to empirically determine the optimal number Keep samples cold at 4 C before sonication to maintain sample integrity Maintain temperature of the samples at 4 C to maintain sample integrity High SDS favours better sonication but inhibits immunoselection optimal range 0 1 to 1 Final SDS concentration should not be higher than 0 15 to 0 20 e g
10. 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 True Micro ChIP kit Conventional ChIP protocols require high numbers of cells hundreds of thousands cells at least limiting the application for ChIP technology to few cell samples Recently ChIP assays on smallest amount of cells have been reported The procedure requires tedious optimization of several reaction conditions to face the increased background observed in ChIP performed with reduced amount of cells That might consequently lead to considerable time and lab expenditures To reduce these tedious steps Diagenode provides the new Auto True MicroChIP kit with optimized reagents and protocol to enable successful ChIP on as few as 10 000 cells Moreover the Auto True MicroChiIP kit protocol has been thoroughly opti
11. the sheared chromatin Preparation Antibody Mix Antibody x ul Beads Wash Buffer tBW1 100 x Antibody coating m Elution buffer Input i ChIP reaction Bead washes Wash eens Elution buffer IP 9 10 11 12 8 gt SS dt pas S ssi Pure Tube Description 200 ul protocol 1 Input ae 2 Empty 3 Magnetic beads 10 ul 4 Beads Wash Buffer tBW1 100 pl 5 Beads Wash Buffer tBW1 100 pl 6 Antibody Mix 100 ul 7 Sheared Chromatin Mix 200 pl 8 Wash Buffer tW1 150pl 9 Wash Buffer tW2 150pl 10 Wash Buffer tW3 150pl 11 Wash Buffer tW4 150pl 12 Elution buffer tE1 Elution buffer tE2 96 ul 4ul This Auto MicroChIP 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 The Input will be prepared in well 1 just before the reverse crosslinking step see instructions in page 19 Innovating Epigenetic Solutions PAGE 25 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 Elution buffer Input
12. 10 in figure 4 H3K4me3 IgG Figure 5 25 0 Average and error bars of 10 IP s performed with IP Star Compact on Human Hela cells using the Diagenode 20 0 mel antibody H3K4me3 GAPDH p E MB2 150 2 m Sat2 Ee 10 0 5 0 0 0 a aE H3K4me3 IgG Innovating Epigenetic Solutions PAGE 31 ChIP sequencing The True MicroChlP kit protocol has been optimised for ChiP seqon an Illumina Next Gen sequencer The recommended amount of starting material for the Illumina sample prep is 10 20 ng of IP d DNA Depending on the cell type the target protein abundance and the antibody used you should recover between 500 pg to a few nanograms of IP DNA when starting with 10 000 cells Moreover after quantification and qPCR analysis you could have only picogram amounts of IMmunoprecipitated DNA left for sequencing Therefore an amplification step is necessary before sequencing the sample using a classical library preparation protocol Thus to provide a complete solution for ChlP sequencing on 10 000 cells Diagenode has developed a library preparation protocol for use with limited quantity of DNA The MicroPlex library preparation kit requires only picogram amounts of ChIP d DNA to start library preparation This kit allows for rapid amplification of few DNA picograms combined with the conversion of DNA into a sequencing ready preparation for the Illumina platform The True MicroChIP kit has been fully validated in ChIP seq in associ
13. 3 hours WAZ 4 DNA decrosslinking and purification 5 hours overnight 253 5 qPCR and data analysis before amplification and sequencing 2 to 3 hours 3 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 4 Place the prepared tube strip on the right cooling heating block of the workstation 9 Close the workstation door and lock it using the following icon E 7 eeeeeee gt ren H LEJ mim e TE si aes Innovating Epigenetic Solutions PAGE 27 6 Press the following icon Pi Select the protocol of interest Press start SK8G V52 Ver0 7 ChIP_Small_Demo HLD RA The program ended successfully diagengy IMPORTANT NOTE If the ChIP protocols do not appear in the screen 1 Open the SX 8V52 directory 2 Open Easy start ini file Write the directory location of the protocols The Easy start ini file should contain the following information EASYSTARTSCREEN HoldFilePath C Documents and Settings Desktop New software protocols ChIP Ab Coating for loading ChIP Direct protocols or HoldFilePath C Documents and Settings Desktop New software protocols ChIP IP and beads incubation for loading ChIP Indirect protocols In red it is indicated the directory location of the ChIP protocols 3 Start now SX 8G V52 software through SX 8G V52 exe file 4 Press button for Easy Protocol
14. D H3K9me3 11 36 A 80 0 ChIP 1 ChIP 2 50 70 of input Auto ChIP 70 0 ChIP 1 56 25 of input 1 26 IgG H3K9me3 SD IgG 0 69 SD H3K9me3 23 84 SD IgG 1 4 SD H3K9me3 2 38 Cc SD IgG 0 17 SD H3K9me3 1 12 ore a SD IgG 0 09 B 98 62 95 26 SD H3K9me3 0 65 D ChIP 1 ChIP 2 43 83 44 75 2 06 1 42 1 54 i 1 42 SD IgG 0 28 SD H3K9me3 1 6 ChIP 1 ChIP 2 57 83 56 25 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 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 www diagenode com diagendtie PAGE 8 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Kit Method Overview Chromatin DNA Shearing Chromatin DNA Preparation Bioruptor Sonication
15. Ea Chromain Ah Bead washes Elution buffer IP Pure Tube Description 200 pl protocol 1 Input dki 2 Empty 3 Magnetic beads 10 ul 4 Beads Wash Buffer tBW1 100 pl 5 Beads Wash Buffer tBW1 100 pl 6 Beads Wash Buffer tBW1 100 pl 7 Sheared Chromatin Mix Antibody 200 pl 8 Wash Buffer tW1 150 pl 9 Wash Buffer tW2 150 pl 10 Wash Buffer tW3 150 ul 11 Wash Buffer tW4 150 pl 12 Elution buffer tE1 Elution buffer tE2 96ul 4ul Auto True MicroChIP 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 The Input will be prepared in well 1 just before the reverse crosslinking step see instructions in page 19 www diagenode com diagendle PAGE 26 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL 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 1 Cell collection and protein DNA crosslinking 1 to 2 hours 1 2 Cell lysis and chromatin shearing 1 hour 1 3 Magnetic immunoprecipitation and washes Overnight
16. P seq data analysis recommendations To find the captured regions of the genome after the 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 built in aligners with default settings worked very well for our ChIP seq 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 C 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 y
17. 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 T Schedule Manager Yer1 0 www diagenode com diagendtie PAGE 28 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Alternatively Incubation time for antibody coating and temperature and incubation time for the IP reaction can be adjusted in an existing protocol by selecting the modify button The modified protocol can also be saved as new protocol SX8G 52 Yer0 7 ChIP_Small_Demo HLD i LA The program ended successfully diagendde Modify Parameter for ChIP x 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 lt 7 The program will run through the following steps magnetic bead washes IP and IP washes Task Manager2 2000 11 13 1 9 2 Volume Mixing 1 9 3 Volume Mixing 1 9 4 Volume Mixing 12 0 Magnet OFF 0 9 6 Magn s z i 1 9 7 Interium Height Z Move 22 tera Heigrtz we During protocol the next window will be displayed indicating the 1 10 2 Action z s s 110 3 Pre asp step that the protocol is processing 1 10 4 Beads resuspend 1 10 5 Pre Dsp 1 10 6 Antibody Mix 0 1 11 1 Action Z 1 11 2 Volume Mixing 1 11 3 Mag
18. T is defined as 200 ul protocol 10 INPUT 20 ul sheared chromatin mix 76 ul Elution buffer tE1 1 INPUT 2 ul sheared chromatin mix 94 ul Elution buffer tE1 NaCl Elution Buffer tE2 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 PAGE 21 CAUTION Screen Caution When the protocol finishes the user can return to the protocol list screen A or warm the Do you want to start again a protocol peltier block screen B to eliminate possible condensation in the block diagenc e CAUTION Temperature unit maintenance is Defined protocol name lists executed diagendte diagencg te CAUTION Temperature unit maintenance x Remaining time gt Right block diageno e Note 1 RNase treatment by incubating the samples with RNase at 37 C during 30 minutes can be performed after the reverse crosslinking and it is recommended for Chlp seq experiments However Diagenode does not provide RNase Note 2 DNA purification using MicroChIP DiaPure columns Cat No C03040001 Al
19. al 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 to change protocol parameters Major benefits of Diagenode Automated Platforms SX 8G IP Star Compact Sx 8G IP Star I High resolution ChIP seq and MeDIP seq profiles Automated library preparation for Next Generation sequencing Reduces hands on time to just 30 minutes Reduces variability between operators and labs Ideal for low sample starting amounts S S 7 s n S Compatible with Diagenode Kits Auto ChIP kit Auto Histone ChIP seq kit Auto Histone ChIP seq kit Auto True Micro ChIP Kit Auto MeDIP kit Auto MethylCap kit Auto hMeDIP Auto IPure kit Reduces cross contamination NV www diagenode com diagendtie DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL E SX 8G IP Star Compact SX 8G IP Star Applications Software User interface User friendly Dispensing Protocol optimization flexible parameters New protocol development Characteristics Innovating Epigenetic Solutions ChIP seq MeDIP seq MethylCap seq hMeDIP IPure Sample preparation Re ChIP MagBisulfite RNA IP Library preparation for NGS platforms Pr
20. and pooled before loading onto agarose gel The protocol for shearing analysis is described in Additional Protocols 3 Antibodies The optimal amount of antibody to use per ChIP experiment has to be optimized for each antibody However we recommend to start with 0 25 ug of antiboby per IP when performing ChIP on 10 000 cells The kit contains a negative IgG and a positive H3K4me3 control antibody We recommend including one IgG negative IP control in each series of ChIP reactions We also recommend using the positive control ChiP seq grade H3K4me3 antibody at least once The kit also contains human qPCR primer pairs for amplification of a positive and negative control target for H3K4me3 GAPDH TSS and Myoglobin exon 2 respectively 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 PAGE 11 www diagenode com diagendtie PAGE 12 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL 5 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 ar
21. ating Epigenetic Solutions 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 seg 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 Browser 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 seg 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 Th
22. ation with the MicroPlex library preparation kit sl a zz JM bce E pis p13 pi22 pli 2 qii l q11 22 q11 23 q21 11 q21 12 q21 3 q221 q223 q31 2 q31 32 q321 q33 q34 035 q36 1 q36 3 Li gal kene p22 2 p21 3 p211 p153 164 kb 8 500 kb 520 kb 10 000 cells reads 10 000 cells peaks 100 000 cells reads 100 000 cells peaks 1 million cells Broad Inst reads aE 1 million cells Broad Inst peaks B R Matched by Broad Institute data set R Unatched by Broad Institute data set 2 Figure 2 A ChIP has been peformed with H3K4me3 antibody amplification of 17 pg of DNA ChIP d from 10 000 cells and amplification of 35 pg of DNA ChIP d from 100 000 cells control experiment The IP d DNA was amplified and transformed into a sequencing ready preparation for the Illumina plateform with the True MicroPlex library preparation kit The library was then analysed on an Illumina Genome Analyzer Cluster generation and sequencing were performed according to the manufacturer s instructions B We observed a perfect match between the top 40 of True MicroChIP peaks and the reference dataset Based on the NIH Encode project criterion ChIP seg results are considered reproducible between an original and reproduced dataset if the top 40 of peaks have at least an 80 overlap ratio with the compared data set www diagenode com diagendtie PAGE 32 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL ChI
23. binding to protein A or protein G Understanding the benefits of using an ultrasonic water bath Buying an ultrasonic bath Determine water bath specifications Optimize the incubation time with an ultrasonic bath Using the kit without an ultrasonic water bath Optimize primer design Include negative and positive controls Troubleshoot high Ct values Determine the ratio between Ct NegCtl and Ct Target Minimize high background Using end point PCR analysis rather than quantitative PCR Samples can be frozen at several steps of the protocol There is a significant difference in affinity of different 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 Protein A Protein G Human IgG1 Fit IgG2 tae IgG3 IgG4 IgGM Use anti Human IgM IgGF IgGA Mouse IgG1 IgG2a TEF IgG2b F Ig63 IgGM Use anti Mouse IgM Rat IgG1 IgG2a lt ET IgG2b ree IgG2c Chicken All Isotypes Cow All Isotypes F Goat All Isotypes lt Guinea Pig All Isotypes Hamster All Isotypes Horse All Isotypes ddd Pig All Isotypes Rabbit All Isotypes Sheep All Isotypes a The use of ultrasonic energy to enhance mass transport across liquid solid interfaces can dramatically accelerate antigen binding to antibo
24. cell range from 10 000 cells to 100 000 cells to allow performing ChIP assay on 10 000 to 100 000 cells Determine the number of cells you would like to use per ChIP reaction between 10 000 and 100 000 cells and perform shearing on that cell number Fixation can be done on larger cell numbers scale accordingly volumes of Lysis Buffer tL1 and HBSS to use and cell lysate will then be split into 100 ul aliquots corresponding to the number of cells that will be use per IP reaction before shearing Then sheared chromatin will be diluted two times with ChIP Buffer tC1 before performing the IMmunoprecipitation 2 Shearing optimization and sheared chromatin analysis Before starting the ChIP the chromatin should be sheared to fragments in the 100 to 600 bp range Our kit is optimized for chromatin shearing using the Bioruptor We recommend using Diagenode s micro tubes as shearing has been shown to be more efficient and reproducible using these tubes The shearing conditions mentioned in the protocol are adequate for a variety of cell types However you should optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP Nevertheless analysis of shearing efficiency is not obvious when working with 10 000 cells due to the low amount of DNA recovered after sonication and crosslinking reversion for subsequent analysis on agarose gels Therefore at least 6 replicates should be performed to check the shearing efficiency
25. col g 1 118 x 10 5 x rx rpm2 where r is the radius www msu edu venkata1 gforce htm It is possible to centrifuge the 1 5 ml tubes at 1 000 2 000 g for 20 seconds Store e 4 C Do not ireeze pAb from rabbit guinea pig pig human IgG MAb from mouse Ig62 human IgG1 2 and 4 and rat Ig62c Some inhibitors are unstable in solution The provided P I mix should be kept frozen at 20 C and thawed before use Add protease inhibitor mix to buffers just before use in HBSS Steps 1 and 2 Lysis Buffer tC1 Step 2 ChIP Buffer tC1 Step 3 Discard within 24 hours Add phosphatase inhibitors or others to Lysis Buffer tC1 and ChIP Buffer tC1 if necessary depending on your research field and protein s of interest Add NaBu for histone ChIPs Use the non immune lgG fraction from the same species the antibodies were produced in as a negative control Incubation with uncoated beads could also be used as a negative ChIP control Use one antibody in ChIP and the same antibody that is blocked with specific peptide To specifically block one antibody pre incubate the antibody with saturating amounts of its epitope specific peptide for about 30 minutes at room temperature before use In the IP incubation mix 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 Antibody antigen recognition can be significant
26. d as follows recovery 100 2 Ctlinput 3 32 Ct sample This equation assumes that the PCR is 100 efficient amplification efficiency 2 For accurate results the real amplification efficiency If known should be used Criteria to decide whether the sample is good enough for sequencing will be largely target dependant Therefore the following are only general guidelines e the recovery of the positive control target should be at least 5 e the ratio of the positive versus the negative control target should be at least 5 Innovating Epigenetic Solutions How to perform Automated ChIP in the SX 8G IP Star Compact SX 8G IP STAR COMPACT www diagenode com di PAGE 14 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL How to perform Automated ChIP in the SX 8G IP Star Compact Chromatin preparation The protocol below is for use with 10 000 cells per ChIP To perform ChIP with higher cell numbers refer to Notes Before Starting STEP 1 Cell collection and DNA protein crosslinking Harvest and count the cells Add medium to cells to a final volume of 1 ml Add 27 ul of 36 5 formaldehyde per 1 ml sample Invert tube and Incubate 10 minutes at RT Add 115 ul of Glycine to the sample Invert the tube and Incubate 5 minutes at RT Work on ice from this point anwards Centrifuge at 300 x g for 10 minutes at 4 C Aspirate the supernatant slowly di Sy E Wash cells with 1 ml ice cold HBSS with inhibit
27. dies the typical rate limiting step in ChIP See http www bransonic com model_3510 asp Branson Cat No CPN 952316 or Fisher Scientific Cat No 15 337 22F Model MT 3510 Capacity 5 5 liters Size LxWxH 29x15x15 cm Frequency 42 kHz Max power requirement 130 W RF Power 130 W Incubation of 15 30 minutes is usually sufficient but may differ depending on antibody target kinetics A longer incubation may be required in some cases Without the bath a long incubation at 4 C should be used Depending on the antibody and target the times of incubation range from 2 to 16 hours and should be determined empirically for each antibody Primer length 18 to 24 nucleotides and primer Tm 60 C 3 0 C GC 50 4 Negative PCR controls PCR with DNA from samples P d with non immune antibodies negative IgG Alternatively PCR using DNA from ChIP samples and primers specific for a DNA region to which your antigen of interest is not binding Positive PCR control PCR using input DNA Use more input chromatin in the case of high Ct values The ratio between target IP and negative control IP depends on the antibody used Keep the antibody binding beads in suspension during the experiment Check by eye that equal pellets of beads are present in each tube Washes step 3 are critical If gel electrophoresis is used to estimate intensities of PCR products the relative occupancy of a factor at a locus is the ratio of the intensit
28. diogendtie Innovating Epigenetic Solutions AUTO True MicroChiP KIT Cat No C01010140 Version 2 03 14 Technical Assistance amp Ordering Information Diagenode s a BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA LIEGE SCIENCE PARK 400 Morris Avenue Suite 101 Rue Bois Saint Jean 3 Denville NJ 07834 USA 4102 Seraing Belgium Tel 1 862 209 4680 Tel 32 4 364 20 50 Fax 1 862 209 4681 Fax 32 4 364 20 51 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 company distributors php For the rest of the world please contact Diagenode s a Contents Tista r se iii irene 4 SX 8G IP Star Automated System for ChIP MeDIP SMBD ee 5 it Te LU aura daddi 8 Kit UNIS 6 cet ang Gh oh he ar ie ath ae ere ed ene hoe oe Re ee E ee ee 9 9 AE SOY 1G E E E E E A PETRI Ri G Required Materials Not Provided i i e 10 Remarks before Starting sas tie ia LARE RA RTLA La eeed 11 How to perform Automated ChIP in the SX 8G IP Star Compact ccc eee ees 13 step I Ceucollectionrand DNA protein CrOSSUNKIN G erates nas ira Pues ARE abd a a HOO SE 13 oep LSI STS andenromaun Shearing serea apaisada ere la i ia PI E pees ded we be KE 13 SIOE RR RETTE 15 How to perform Automated ChIP in the SX 8G IP Star i 22 Step 1 Cell collection and DNA p
29. e 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 pl 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 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 Required Materials Not Provided Reagents e Gloves to wear at all steps e Phosphate buffered saline PBS e Cell culture medium e 1 M Sodium butyrate NaBu Cat No kch 817 001 optional e Trypsin EDIA e Formaldehyde fresh MolBiol Grade e Hank s balanced salt solution HBSS e Ethanol e Phenol chloroform isoamyl alcohol 25 24 1 e qPCR reagents e Quant IT dsDNA HS assay kit Invitrogen e JE Equipment and accessories e DiaMag 1 5 magnetic rack Cat No kch 816 015 e Refrigerated centrifuge for 1 5 ml tubes
30. e usually not sufficiently sensitive In most cases It Is sufficient to use approximately one quarter of the IP d material for quantification when working with 10 000 cells 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 6 Quantitative PCR Before sequencing the samples we recommend analysing the IP d DNA by qPCR using at least 1 positive and 1 negative control target In order to have sufficient DNA left for sequencing we recommend not using more than one third of the total IP d DNA for qPCR You can dilute the DNA 1 4 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 7 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 recovery 100 2 Ctlinput log X log2 Ct sample e Ct sample and Ct input are threshold values obtained from exponential phase of qPCR for the IP d DNA sample and input sample respectively e logx log2 accounts for the dilution 1 x of the input If the amount used for the input was 10 of the amount used for ChIP the recovery can be calculate
31. ecutable Buttons e The user presses the Back button The user returns to the Protocols screen e The user presses the Shutdown button The screen shall be changed to Power Off e The 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 The user presses the Next button Then screen shall be changed to Configuration or Layout information PAGE 17 DIRECT ChIP INDIRECT ChIP Configuration Configuration peha Mixing time Temperature Mix Speed Mixing time Temperature Mix Speed Ab coating k a IP reaction Cc IP reaction C Beads incubation L Washes i diagencte W Ne diageng e Screen Configuration After the user presses the next button from the Sample number screen the Configuration screen appears Buttons e The user presses the Back button The user returns to the Protocol List screen e The user presses the Next button The screen shall be changed to Layout information Keyboard e The user presses the Save Parameter button The screen will Confirmation be chang
32. ed 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 C No Returns the user to the display of the Configuration k screen e The user presses the Text box The screen will be changed to Keyboard or Speed list menu Speed list menu www diagenode com diagendtie PAGE 18 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Layout information Screen Layout Information After the user presses the next button from Sample number screen or Configuration screen the Layout Information screen appears Buttons e The user presses the Back button The user returns to the previous screen e The user presses the Next button The screen shall be changed to Set confirmation e 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 e Based on the selected protocol the user follows the indications provided in the screens to set up correctly the different reagents and samples MIMyel Mie Block Tip Screen Layout Information Reagent information Av DIB Elution butter Beads Wash Buffer Beads Wash Buffer tEW1 Elution Buffer Elution Buffer tE1 IP wash 1 Wash Buffer tW1 IP Was
33. es with RNase at 37 C during 30 minutes can be performed after the reverse crosslinking Diagenode does not provide RNase 9 DNA purification DNA purification using MicroChIP DiaPure columns Cat No C03040001 Alternatively phenol chloform extraction can be performed 1 Ina 1 5 ml microcentrifuge tube add 5 volumes of ChIP DNA Binding buffer to each volume of sample 100 ul sample 500 ul buffer Mix briefly Transfer mixture to a provided Spin column in a Collection tube Centrifuge at 10 000 x g for 30 seconds Discard the flow through Add 200 ul DNA Wash buffer to the column Centrifuge at 10 000 x g for 30 seconds Repeat wash step Add 6 100 ul DNA Elution buffer directly to the column matrix Transfer the column to a new 1 5 ml microcentrifuge tube and centrifuge at 10 000 x g for 30 seconds to elute the DNA or Fe W N 6 That corresponds to the purified immunoprecipitated 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 PAGE 29 www diagenode com diagendtie PAGE 30 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Quantitative PCR amp Data Analysis Before sequencing the samples we recommend analysing the IP d DNA by qPCR using at least 1 positive and 1 negative control targ
34. et In order to have sufficient DNA left for quantification and sequencing we recommand to use one third of the total immunoprecipitated DNA for qPCR analysis 1 Prepare the gPCR mix using SYBR Green master mix qPCR mix total volume of 25 ul reaction e 1 ul of primer pair stock 5 uM each reverse and forward e 12 5 ul of master mix e g iQ SYBR Green supermix e 5 0 ul of purified diluted DNA sample and purified input s e 6 9 ul of water Use the following PCR program 3 to 10 minutes denaturation step at 95 C please check carefully supplier s recommendations about Taq polymerase activation time followed by 45 cycles of 30 seconds at 95 C 30 seconds at 60 C and 30 seconds at 72 C These conditions may require optimisation depending on the type of Master Mix or gPCR system used s Figure 4 m EIF4A2p ChIP was performed with IP Star m GAPDH p Compact on human HeLa cells using the siii MB2 Diagenode antibody H3K4me3 Cat No i sui pAb 003 050 Sheared chromatin from 10 000 cells 0 25 ug of the H3K4me3 antibody and 0 25 ug of the negative IgG control 3 were used per IP Quantitative PCR was x performed with the positive controls 10 0 GAPDH TSS and EIF4A2 promoter and the negative controls Myoglobin exon 2 and Sat 2 primer sets The recovery expressed as a of input the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis is shown IP1 IP2 IP3 IP4 IPS IP 6 IP7 IP 8 IP9 IP
35. ffer tL1 1 ml 4 C Protease inhibitor cocktail 200x PIC 270 pl 20 C ChIP Buffer tC1 omil 4 C Beads Wash Buffer tBW1 15 ml 4G Protein A coated magnetic beads 220 pl Do ni Wash Buffer tW1 5 ml 4 C Wash Buffer tW2 gml 4 G Wash Buffer tW3 5 ml 4 C Wash Buffer tW4 gml 4 C Elution Buffer tE1 12 ml 4 C Elution Buffer tE2 500 pl 4 C Precipitant tP1 1 2 ml 4 C Co precipitant tCP1 60 ul 20 C Co precipitant tCP2 60 ul 20 C Control IgG 10 ul 20 C ChIP seq grade antibody H3K4me3 10 ug 20 C ChIP seq grade GAPDH TSS primer pair 50 pl 20 C ChIP seq grade Myoglobin exon 2 primer pair 50 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 paramagnetic beads kch 802 220 The beads are supplied for 16 IPs 220 pl 4 C kch 802 600 detergent and 0 02 sodium azide 660 ul Do not freeze kch 802 150 included 1500 pl Protein G coated paramagnetic beads kch 818 220 The beads are supplied for 16 IPs 220 pl 4 C kch 818 600 detergent and 0 02 sodium azide 660 ul Do not freeze kch 818 150 included 1500 ul Rabbit IgG kch 504 250 1 ug ul 250 ul 4 C Mouse IgG kch 819 015 1 ug yl 15 ul 4 C Antibodies www diagenode com Primer pairs BUM each Rv amp Fw www diagenode com www diagenode com diagendle PAGE 10 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Tabl
36. h 2 Wash Buffer tW2 IP wash 3 Wash Buffer tW3 IP wash 4 Wash Buffer tW4 C F IP wash No 1 4 Block Regent Tip Rack DIRECT ChIP INDIRECT ChIP 1200000000 PCR tube 100000000 PCR tube 11090000000 1100000000 10 F A Well 7 ZAR 100 P F ry Well 7 ari 900000000 900000000 i 80600000000 Well 6 an piles 00000000 Well 3 Magnetic beads 700000000 nl 00000000 60600000000 Well 3 Magnetic beads 00000000 560000000 00000000 1060000000 00000000 300000000 00000000 2060000000 00000000 100000000 100000000 Well 7 Sample 200 ul sheared Chromatin mix Well 6 Ab in buffer 100 ul Bead Wash buffer tBW1 x ul Ab Well 3 Magnetic beads 10 ul Note that Diagenode protein A and proteina G magentic beads have a binding capacity of 3 ug antibody 10 ul beads Innovating Epigenetic Solutions PAGE 19 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 Rightblock Left block C Right block C Back diagengde diageno e 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
37. hod 1 Direct method Ab Beads gt Ag 1 Beads pre wash 7 2 Antibody coating I i 3 IP reaction S 4 IP washes 5 Elution 2 Indirect method Ag Ab gt Beads 1 Beads pre wash 2 IP reaction 3 Beads incubation 4 IP washes 5 Elution diagenotie Diagenode Splash Screen A0 After the software start up screen disappears PAGE 15 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 www diagenode com diagendtie PAGE 16 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Defined protocol name lists diagendtie Sample number Sample number e feTe ttlcisl number Keyboard Innovating Epigenetic Solutions Screen Categories Name Protocol List After the user presses the Categories Namel button the Categories Namel appears When selected the protocol on the protocol list the Run button shall turn ex
38. lly remove the supernatant and add 500 ul of ice cold 70 ethanol to the pellet 14 Centrifuge for 10 minutes at 14 000 x g 13 000 rpm at 4 C Carefully remove the supernatant leave tubes opened for 30 minutes at RT to evaporate the remaining ethanol 15 Resuspend the pellet in 10 ul of TE That corresponds to the purified DNA from the sheared chromatin Several DNA pellets can be pooled at this step to have DNA corresponding to a minimum of 60 000 cells in 10 ul of TE 16 Run samples 10 ul of DNA 2 ul of 6x loading dye in a 1 5 agarose gel along with DNA size marker to visualise shearing efficiency 1 Figure 3 Hela cells were fixed with 1 formaldehyde for 10 minutes at RT Cell lysis are performed using the Lysis Buffer tL1 of the Diagenode True MicroChlIP kit Samples corresponding to 10 000 cells are sheared during 5 rounds of 5 cycles of 30 seconds ON 30 seconds OFF with the Bioruptor Plus combined with the Bioruptor Water cooler Cat No BioAcc cool at HIGH power setting position H For optimal results samples are vortexed before and after performing 5 sonication cycles followed by a short centrifugation at 4 C 10 ul of DNA equivalent to 60 000 cells are analysed on a 1 5 agarose gel lane 1 lane M 100 bp DNA Molecular Weight Marker www diagenode com diagendtie PAGE 36 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Troubleshooting guide Crosslinking and fixation Cell lysis
39. ly affected by crosslinking resulting in loss of epitope accessibility and or recognition Use ChIP grade antibodies or several antibodies directed against different epitopes of the same protein Verify that the antibodies work directly in IP on fresh cell extracts When testing new antibodies include known ChIP grade antibodies as a positive control 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 Empirically determine amount of target and antibody Titration of the antibody is recommanded to find the optimal concentration When working with 10 000 cells start with 0 1 to 0 5 ug of antibody per ChIP On higher amounts of cells use 1 2 ug of purified antibody per ChIP for abundant proteins like histones Efficient IPs result from optimal ratios between the amount of chromatin and the amount of antibody More antibody or less chromatin can be required with low affinity to antigen or high abundance of target protein e g histones Insufficient amount of antibody can result in low efficiency of ChIP whereas large excess of antibody might lead to lower specificity www diagenode com diagendtie Immunoselection incubation PCR tips Sample storage and freezing Innovating Epigenetic Solutions DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Concerns about antibodies
40. mised by Diagenode for ChIP followed by high throughput sequencing on Illumina Next Gen sequencers To allow the generation of consistent results in ChIP seg on 10 000 cells Diagenode has also optimised a library preparation protocol on limited amount of immunoprecipitated DNA The new MicroPLEX library preparation kit allows the preparation of libraries for sequencing on picogram amount of immunoprecipitated DNA Thus association of the Auto True MicroChlP kit with the MicroPLEX library preparation kit provides optimised solutions to perform ChIP sequencing on limited amount of cells 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 SEGUE The IP Star reduces our processing time down from one day of manual work to just one overnight Feedback run with only 30 minutes of hands on work The IP Star has made all our ChIPs consistent and the process completely reliable regardless of the operator or the time of day Dr John Lambourne Postdoctorate Researcher at the Innovation Centre McGill University Canada Innovating Epigenetic Solutions 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 yourlab s productivity efficiency and experiment
41. ml ice cold HBSS with inhibitors Invert the tube to resuspend the cells and centrifuge at 300x g for 10 minutes at 4 C 8 Aspirate the supernatant and keep the cell pellet on ice STEP 2 Cell lysis and chromatin shearing 9 Add 25 ul of complete Lysis Buffer tL1 Lysis Buffer tL1 Protease Inhibitor Cocktail PIC per 10 000 cells and agitate manually the bottom of the tube to resuspend the cells 10 Incubate on ice for 5 minutes 11 Add 75 ul of complete HBSS HBSS PIC per 10 000 cells and sonicate aliquots of 10 000 cells in 100 ul for 1 to 5 x 5 cycles of 30 seconds ON 30 seconds OFF using the Bioruptor Optimization is needed depending on the cell type and the Bioruptor R model used 12 Centrifuge at 14 000 x g for 10 minutes and collect the supernatant 13 Add 120 ul of complete ChIP Buffer tC1 ChIP Buffer tC1 PIC to 100 ul sheared chromatin This is the Sheared chromatin mix e Use 200 ul for the IP e Keep 20 ul as input Note Diluted Chromatin is ready now for the IP Select automated ChIP IPure 200 ul protocols indirect method in the IP Star system www diagenode com diagendtie PAGE 24 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL STEP 3 Dispense prepared reagents into the corresponding tubes see picture below ChIP Direct Method Ab coating With this method the antibody is first coated on the surface of the magnetic beads and after that the bound antibodies are added to
42. net ON 1 11 4 Volume Mixing 1 11 5 Air disp 1 11 68 Magnet OFF 0 1 11 7 Air Dsp 1 11 8 Interium Height Z Move x l 1 12 1 0 f Wait msec 5000 wait time msec _ 1 12 2 IP reaction 0 Stack DUP 1 12 3 10 Lig_in POP I P_SPEED_HtAsp Speed 1 13 1 Right block Well6 Wait_msec 200 waittime msec 1 13 2 Action Z Stack DUP 1 13 3 Beads resuspend 1 13 4 IP reaction 0 Lig_out POP P_SPEED_H Disp Speed Pass_time 1 13 5 Air Dsp _ IF_Goto LE 7200 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 amp Yell6 1 14 3 Action Z 1 14 4 Beads resuspend Innovating Epigenetic Solutions 8 ChIP IPure After the IP washes the following window will be appear Attention Please Open the door Add zul input in well 1 Ang put the cap on the PCR tube P lt lt lt CAUTION gt gt gt gt D e 1 Prepare input as a Add 10 Input to well 1 gt 20 ul of input 76 ul Elution Buffer Et1 4 ul elution buffer Et2 b Add 1 Input in well 1 2 ul input 94 ul Elution Buffer Et1 4 ul Elution Buffer Et2 2 Close the tube strip with the corresponding caps 3 Press OK oe 10 or 1 Input mae Ra ama ew air 4 Reverse crosslinking will be performed at 65 C for 4 hours or O N Note Optional RNase treatment by incubating the sampl
43. ors Invert the tube to resuspend the cells and centrifuge at 300 x g for 10 minutes at 4 C 8 Aspirate the supernatant and keep the cell pellet on ice STEP 2 Cell lysis and chromatin shearing 9 Add 25 ul of complete Lysis Buffer tL1 Lysis Buffer tL1 Protease Inhibitor Cocktail PIC per 10 000 cells and agitate manually the bottom of the tube to resuspend the cells 10 Incubate on ice for 5 minutes 11 Add 75 ul of complete HBSS HBSS PIC per 10 000 cells and sonicate aliquots of 10 000 cells in 100 ul for 1 to 5 x 5 cycles of 30 seconds ON 30 seconds OFF using the Bioruptor Optimization is needed depending on the cell type and the Bioruptor model used 12 Centrifuge at 14 000 x g for 10 minutes and collect the supernatant 13 Add 120 ul of complete ChIP Buffer tC1 ChIP Buffer tC1 PIC to 100 ul sheared chromatin This is the Sheared chromatin mix e Use 200 ul for the IP e Keep 20 ul as input Note Diluted Chromatin is ready now for the IP Select automated ChIP IPure 200 ul protocols indirect method in the IP Star Compact system Innovating Epigenetic Solutions Running a protocol Automated Epigenetic Applications on Magtration Technology Genetein software ver 1 0 Copyright 2010 diagenc e Protocols Maintenance Information diagende Protocols ve x e I A a MM hMeDIP MethylCap eS Ss a Re ChIP MagBisulfite RNA IP dioagendiie ChIP preparation met
44. otocols ACA ZAT ma Me a pA MeDIP MethylCap A lt i MAST lt as L Re ChIP diageng 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 Dx 610H 100 kg 8 Nozzles X Y Z 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 Z axis 4 95 C Improved reproducibility 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 and 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 S
45. our 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 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 MACS2 e SI CER 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 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
46. rior to immunoselection Gel analysis of sheared chromatin Antibody bead binding Protease inhibitors and other inhibitors Negative ChIP controls Antibody in IP Load enough DNA on gel Use correct agarose concentration Use correct running buffer concentration and run time Make sure beads are in suspension Use proper bead centrifugation methods Store beads at 4 C Determine antibody binding capacity Store protease inhibitors properly Always use fresh complete buffers Use other inhibitors as needed Use non immune IgG in the IP incubation mix Do not add antibody to the IP to serve as a negative control Use an unblocked antibody and specifically blocked antibody in parallel Determine number of negative controls needed Antibody antigen recognition may affect ChIP Use ChIP grade antibodies include controls and test antibodies before ChIP Determine amount of antibody per ChIP PAGE 37 Chromatin equivalent to at least 60 000 cells can be be visualized on a gel Do not use an excessive amount or it will obscure the visualization The DNA amount to load depends on well size and on the gel size Use a 1 1 5 agarose gel 1x TAE or TBE is preferred to 0 5x TAE which can lead to smears Run slowly The provided beads are coated with protein A Resuspend into a uniform suspension before each use Use gentle centrifugation 500 x g for 2 3 minutes as described in the manual proto
47. rotein crosslinking nannaa annann aaa Zd olp Lel ysis and enomaun shearing cueas polpa lenta i vous Eea ee RR R a 23 Step 3 Dispense prepared reagents into the corresponding tubes 0 02 24 OAT and aiaa Prol CO CARRO RE 26 muting down the SA 0 0 Peoia esre rn n Eee 29 Quantitative PCR amp Data analysis e e i pae 30 ChiP Seq data analysis recormmendatigns i e ea 32 Adona UPTO O aen E cored r ira E a ana 34 Trou bleshoonng GUNE 4 6 6 X 6 06 E KR eater TR Ali 36 Technical ASSISTA Nigro lapidea eta aaa 37 Ordering INfOrMallon lt 22 sissi iter Back Cover PAGE 3 www diagenode com diagendtie PAGE 4 DIAGENODE AUTO TRUE MICROChIP 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 are 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
48. roughout 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 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 avadis ngs com e Geneious http www geneious com web geneious geneious pro e GenoMiner http www astridbio com genominer html e GenoMatix http www genomatix de PAGE 33 www diagenode com diagendtie PAGE 34 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Aditional Protocols Sheared chromatin analysis This protocol refers to the Diagenode s Elution module Cat No mc magme 002 that can be ordered separately Reagents not supplied gt e RNase cocktail e g Ambion AM 2286 A e Phenol chloroform isoamyl alcohol 25 24 1 e Chloroform isoamyl alcohol 24 1 e 100 Ethanol e 70 Ethanol e Agarose and TAE buffer e TE Take an aliquot of 100 ul of sheared chromatin and spin the chromatin at 14 000 x g 13 000 rpm for 10 min at 4 C Transfer the supernatant to a new tube for chromatin analysis A minimum of 60 000 cells is needed to be vizualized onto agarose gel If each
49. ternatively phenol chloform extraction can be performed 1 Ina 1 5 ml microcentrifuge tube add 5 volumes of ChIP DNA Binding buffer to each volume of sample 100 pl sample 500 ul buffer Mix briefly 2 Transfer mixture to a provided Spin column in a Collection tube 3 Centrifuge at 10 000 x g for 30 seconds Discard the flow through 4 Add 200 ul DNA Wash buffer to the column Centrifuge at 10 000 x g for 30 seconds Repeat wash step 5 Add 6 100 ul DNA Elution buffer directly to the column matrix Transfer the column to a new 1 9 ml microcentrifuge tube and centrifuge at 10 000 x g for 30 seconds to elute the DNA 6 That corresponds to the purified immunoprecipitated DNA www diagenode com diagendtie How to perform Automated ChIP in the SX 8G IP Star SX 8G IP STAR How to perform Automated ChIP in the SX 8G IP Star The protocol below is for use with 10 000 cells per ChIP To perform ChIP with higher cell numbers refer to Notes Before Starting STEP 1 Cell collection and DNA protein crosslinking Harvest and count the cells Add medium to cells to a final volume of 1 ml Add 27 ul of 36 9 formaldehyde per 1 ml sample Invert tube and incubate 10 minutes at RT Add 115 ul of Glycine to the sample Invert the tube and Incubate 5 minutes at RT Work on ice from this point anwards Centrifuge at 300 x g for 10 minutes at 4 C Aspirate the supernatant slowly oe da DD a Wash cells with 1
50. 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 z Remaining time Current Temperature Value diagenctle Screen Running After the user presses the Run button in the Set confirmation screen the Running screen appears Buttons e The user presses the Stop button Then screen shall be changes to Stop Dialog Status screen is preferred as a progress bar that moves across the screen as the step progresses www diagenode com diagendtie PAGE 20 DIAGENODE AUTO TRUE MICROChIP KIT USER MANUAL Running status diagend e ChIP IPure CAUTION e Add input sample in well 1 and on Duten 4 ul 5M NaCl in wells 1 and 12 e After that put caps on tubes diageno e Running status Z Remaining time Right block 5 diagenotie Finish e Open the door e Recover the samples and remove the consumables 1789799998 9 100000000 2 Input If you set dioagendie Innovating Epigenetic Solutions 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 INPU
51. y of the target IP band to the negative control IP band Snap freeze and thaw on ice e g fixed cell pellets and sheared chromatin Pellets of formaldehyde fixed cells can be stored at 80 C for at least a year Sheared chromatin can be stored at 80 C for months depending on the protein of interest Purified DNA from ChIP and input samples can be stored at 20 C for months Avoid multiple freeze thawing 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 Ordering information
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