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1. 41 Store the DNA at 20 C until you are ready to analyze it with qPCR or by high throughput sequencing www diagenode com diagenctie PAGE 16 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL STEP 5 Quantitative PCR analysis 000o Before sequencing the samples we recommend analysing 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 seq grade antibody in SYBR Green qPCR assay using the protocol described below Use your own method of choice for analysing the appropriate control targets for your antibodies of interest 42 Prepare the qPCR mix as follows 20 ul reaction volume using the provided control primer pairs e 10 ul of a 2x SYBR Green qPCR master mix e 1 ulof primer mix e A4ylof water e byulIP dor input DNA 43 Use the following PCR program 3 to 10 min denaturation step at 95 C please check carefully supplier s recommendations about 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 conditions may require optimization depending on the type of Master Mix or qPCR system used ChlP sequencing The iDeal protocol has been optimized for ChIP seq on an lllumina HiSeq Next Gen sequencer However other se2. Resuspend the pelleted beads using the pipet and make sure that you drop them on the bottom of the tube STEP 5 Quantitative PCR analysis 000o 40 Spin the tubes and place them into the DiaMag0 2 magnetic rack wait 1 min and transfer the supernatants into a new labelled 1 5 ml tube and discard the beads 41 Prepare the qPCR mix as follows 20 ul reaction volume using the provided control primer pairs 10 pl of a 2x SYBR Green qPCR master mix 1 pl of primer mix 4 ul of water 5 uL IP d or input DNA 42 Use the following PCR program 3 to 10 min denaturation step at 95 C please check carefully supplier s recommendations about 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 conditions may require optimization depending on the type of Master Mix or qPCR system used www diagenode com diagenctie PAGE 12 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Detailed 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 T175 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 C01019027 in addition to the formaldehyde fixation
3. 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 ChIP sequencing STEP 2 Cell lysis and chromatin shearing KEX 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 rpm and 4 C for 5 minutes and discard the supernatant Keep everything at 4 C from now on 7b Add
4. living cells are first fixed with a reversible cross linking agent to stabilize protein DNA interactions The most widely used reagent to fix cells is formaldehyde which generates covalent bonds between amino or imino groups of proteins and nucleic acids Formaldehyde treatment crosslinks both DNA protein as well as protein protein complexes Following cross linking chromatin needs to be sheared very efficiently into homogeneous small fragments that can subsequently be used in immunoprecipitation IP The Bioruptor shearing devices from Diagenode provide you with high quality sheared chromatin ready to ChIP Currently the Bioruptor is the most widely used and the most cited chromatin fragmentation system as evidenced by more than 1000 publications Shearing may also be accomplished with shearing kits from Diagenode which enable an easy and highly reproducible shearing process for any cell type After fragmentation the sheared chromatin is precipitated with a specific antibody AB directed against the protein of interest The chromatin AB complex is isolated using magnetic beads Finally the precipitated DNA fragments are released from the AB and analyzed Enrichment of specific sequences in the precipitated IP d DNA indicates that these sequences were associated with the protein of interest in vivo Analyzis of specific regions can be performed by quantitative polymerase chain reaction qPCR In recent years ChIP combined with high throughput Next
5. or 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 tis essential to quench the ormaldehyde 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 Add the protease inhibitors to the lysis buffer immediately before use occur Cell type Kit protocol validation HeLa NCCIT 293T Chondrocytes P19 ASC adipose stem cells and Kerationocytes 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 settings of the sonicator appara
6. Generation sequencing ChIP seq has become the gold standard for whole genome mapping of protein DNA interactions Although ChIP seq is a powerful tool the procedure requires tedious optimization of several reaction conditions that might lead to considerable time and lab expenditures To reduce these tedious steps Diagenode provides kits with optimized reagents and protocols for ChIP which enable successful ChIP seq The new iDeal ChIP seq kit protocol has been thoroughly optimized by Diagenode for ChIP followed by high throughput sequencing on lllumina HiSeq Diagenode also offers a wide range of additional tools and products for ChIP analysis including the Bioruptor for chromatin shearing and nucleic acid fragmentation the IP Star Compact for automated ChIP and DNA methylation analysis magnetic racks for 0 2 or 1 5 ml tubes which enable you to work with magnetic beads under optimal conditions 49C and ChIP and ChIP seq grade antibodies against many epigenetic targets In addition Diagenode provides individual reagents such as magnetic beads negative IP controls mouse and rabbit IgG protease inhibitors and deacetylase inhibitors sodium butyrate Diagenode also offers several qPCR primer pairs for the analysis of IP d DNA Peptides can additionally be purchased for use in blocking experiments negative ChIP controls All our products have been extensively validated in ChIP on various targets The combination of all our quality co
7. Representative images made during bioinformatics analysis of ChIP 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 Innovating Epigenetic Solutions PAGE 19 Aditional protocols Sheared chromatin analysis Reagents not supplied with the iDeal ChIP seq kit RNase cocktail e g Ambion AM 2286 A e Phenol chloroform isoamyl alcohol 25 24 1 Chloroform isoamyl alcohol 24 1 e 100 Ethanol 70 Ethanol DNA precipitant Cat No C03030002 DNA co precipitant Cat No C03030001 1 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 1pl of cocktail in 150 ul of ChIP seq 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 iE2 mix thoroughly Incubate samples at
8. TAE which can lead to smears on agarose gel Sheared Pow mueheheared chrormatin dai Most of the sheared chromatin is to be used in the ChIP experiment but remember chromatin 3 hat some of the sheared chromatin is needed as control as it corresponds to the input amounts need to prepare sample for the ChIP experiment and it can also be checked on agarose gel www diagenode com PAGE 21 diagenctie PAGE 22 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Antibody binding beads Beads are in suspension The provided beads are coated with protein A Resuspend into a uniform suspension before each use Bead storage Store at 4 C Do not freeze Antibody binding capacity 10 pg 30 ul 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 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 ncubation with beads which were not coated with antibodies antibodies could also be used as a negative ChIP control control s Use one antibody in ChIP and the same antibody that is blocked with specific peptide Use a specifically blocked antibody in To specifically block an antibody pre incubate the an
9. diagenode com diagenctie PAGE 8 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL 7 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 The kit contains a positive and negative control primer pair which can be used for the CTCF positive control antibody in SYBR Green qPCR 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 1096 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 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 If the amount used for the input was 196 of the amount used for ChIP the recovery can be calculated as follows recovery 2 Ct Ct input sample Ct ampe and Ct are the threshold cycles from the exponential phase of the qPCR for the IP d DNA sample and input respectiv
10. pl to each IP and input sample e Keep IPure beads v2 in liquid suspension during storage at 4 C and at all handling steps as drying will result in reduced performance e The final volume is now 116 ul per IPure reaction Incubate IP and input samples for 10 minutes at room temperature on a rotating wheel 40 rpm Prepare the Wash buffer 1 containing 50 isopropanol Wash buffer 1 24 rxns 100 rxns Wash buffer 1 w o isopropanol 2ml 8ml Isopropanol 10096 2ml 8ml Total volume 4ml 16 ml 34 35 Briefly spin the tubes place in the DiaMag02 wait 1 min and discard the buffer Add 100 ul Wash buffer 1 per tube Close the tubes and vortex well until the beads pellet is completely broken In order to avoid the beads pellet to be too difficult to break down do not let the beads for too long on the magnet incubate for 30 seconds at room temperature on a rotating wheel 40 rpm Never leave the bottle open to avoid evaporation e Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the Diagenode Magnetic Rack Prepare the Wash buffer 2 containing 50 isopropanol as follows Innovating Epigenetic Solutions Wash buffer 2 24 rxns 100 rxns Wash buffer 2 w o isopropanol 2ml 8ml Isopropanol 100 2 ml 8 ml Total volume 4 ml 16 ml PAGE 15 e Never leave the bottle open to avoid evaporation 36 Wash t
11. supernatant Wash the beads with Wash buffer iW1 To wash the beads add 350 ul of iW1 gently shake the tubes to resuspend the beads and incubate for 5 minutes on a rotating wheel at 4 C 25 Repeat the wash as described above once with Wash buffer iW2 iW3 and iW4 respectively STEP 4 Elution decross linking and DNA purification 26 After removing the last wash buffer add 100 ul of Elution buffer iE1 to the beads and incubate for 30 min on a rotating wheel at room temperature Resuspend the beads pellet and transfer it into a new 200 ul tube 27 Briefly spin the tubes and place them into the Diagenode magnetic rack Transfer the supernatant to a new tube and add 4 ul of Elution buffer iE2 Also add 97 5 pl buffer iE1 and 4 ul of buffer iE2 to the 2 5 ul input sample Incubate for 4 hours or overnight at 65 C 28 Pool samples if necessary NOTE Up to 2 samples can be easily pooled If more than 2 samples need to be pooled process each sample purification individually pool final eluates at the end of the IPure purification and concentrate e g using Microcon 29 30 31 32 33 Add 2 ul of carrier to each IP and input sample Add 100 ul of 10096 isopropanol to each IP and input sample Following the addition of isopropanol the solution may become cloudy This is not detrimental to your experiment and will not influence the quality or quantity of your purified DNA Resuspend the provided IPure beads v2 and transfer 10
12. 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 seq 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 ChlP seq please always keep an eye on sequencing quality and the performance of the software
13. 1 5 ml 15 ml and 50 ml tubes Cell counter DiaMag Rotator Rotating wheel Cat No B05000001 Vortex Thermomixer Qubit system Invitrogen qPCR cycler For tissues Petri dishes Innovating Epigenetic Solutions PAGE 7 Remarks before starting 1 Cell number This protocol has been optimized for ChIP on 4 000 000 cells in 350 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 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 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 transcription 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 opt
14. 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 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 uland 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 Bio
15. 5 ml 20 C Rabbit IgG 10 ug 1 ug uU 40 ug 1 ug uU 20 C ChIP seq grade CTCF antibody 10 ug 2 5 ug uU 40 ug 2 5 ug ul 20 C ChIP seq grade H19 imprinting control region primer pair 1004 500 u 20 C ChIP seq grade Myoglobin exon 2 primer pair 100 p 500 u 20 C Carrier 80 ul 320 p 20 C Glycine 10m 40m 4 C Shearing buffer iS1b 7 3m 31m 4 C DiaMag Protein A coated magnetic beads 870 u 3 3 ml 4 C DO NOT FREEZE Wash buffer iW1 0m 42m 4 C Wash buffer iW2 Om 42m 4 C Wash buffer iW3 Om 42m 4 C Wash buffer iW4 Om 42m 4 C ChIP seq grade water 30m 120 ml 4 C Elution buffer iE2 754 720 u 4 C Fixation buffer 6 1m 26m 4 C Wash buffer 1 w o iso propanol 2 ml 8 ml 4 C Wash buffer 2 w o iso propanol 2 ml 8 ml 4 C Buffer C 2ml 8ml 4 C IPure Beads v2 385 ul 1 6 ml 4 C DO NOT FREEZE Elution buffer iE1 4 ml 16 ml 4 C 5x ChIP buffer iC1b 7 5 ml 32 ml 4 C Lysis buffer iL1b 110 ml 470 ml 4 C Lysis buffer iL2 66 ml 280 ml 4 C www diagenode com PAGE 5 diagenctie PAGE 6 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Store DiaMag Protein A coated magnetic beads and IPure Magnetic beads at 4 C Do NOT freeze magnetic beads because they may become damaged Keep the beads in liquid suspension during storage as drying will result in reduced performance Table 3 Modules available separately Description Reference Quantity Chromatin Shearing Optimization Kit Low SDS for T
16. 65 C for 4h or overnight 9 o Os Y do Os 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 9 Centrifuge for 2 min at 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 sample at RT for 10 min on a rotating wheel 11 Centrifuge for 2 min at 13 000 rpm at RT Transfer the top aqueous phase into a new 1 5 ml tube 12 Precipitate the DNA by adding 10 pl DNA precipitant 5 ul of co precipitant and 500 ul of cold 100 ethanol to the sample Incubate at 80 C for 30 min 13 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 14 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 15 Re suspended the pellet in 20 ul of TE buffer 16 Run samples 20 ul of DNA 4 ul of 6x loading dye in a 1 5 agarose gel www diagenode com diagenctie PAGE 20 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL 300 200 100 0 1000 2000 3000 A C Figure 6 Superior chromatin shearing results with the Bioruptor Plus using buffers and protocol of the Diagenode iDeal ChIP seq kit Hela cells were fixed with 196 formaldeh
17. Fs C01020013 25 rxns ChIP Cross link Gold C01019027 600 ul IPure kit v2 x24 C03010014 24 rxns IPure kit v2 x100 C03010015 100 rxns Required materials not provided Reagents Gloves to wear at all steps Formaldehyde 3796 Molecular Grade Phosphate buffered saline PBS buffer 1 M Sodium butyrate NaBu Cat No C12020010 optional 10096 isopropanol RNase DNase free 1 5 ml tubes qPCR SYBR Green Mastermix Reagents for library preparation cluster generation Illumina or ePCR lon Torrent PGM and sequencing Quant IT dsDNA HS assay kit Invitrogen Diagenode ChIP cross link Gold Cat N C01019027 Optional For library preparation we highly recommend High Resolution Library Preparation kit Cat No C05010023 MicroPlex Library Preparation kit v2 Cat No C05010012 12 reactions 12 indices Cat No C05010013 48 reactions 12 indices Equipment Optional ChlPettor System for Transcription Factors Cat No C01010163 Diagenode DiaMag1 5 magnetic rack Cat No B04000003 and Diagende DiaMag02 magnetic rack Cat No B04000001 Diagenode Bioruptor sonication device Cat No Standard B01010001 Plus B01020001 and Pico B01060001 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 Refrigerated centrifuge for
18. ating wheel Add 250 ul sheared chromatin Keep 2 5 pul chromatin aside to serve as input Incubate overnight at 4 C on a rotating wheel The next day briefly spin the tubes place them in the ice cold magnetic rack and discard the supernatant Add 350 pl ice cold Wash buffer iW1 and incubate for 5 min at 4 C on a rotating wheel Discard the wash buffer using the Diagenode magnetic rack Repeat step 24 and 25 once with Wash buffer iW2 iW3 and iW4 respectively STEP 4 Elution decross linking and DNA purification 26 27 28 29 30 31 32 33 After removing the last wash buffer add 100 ul of Elution buffer iE1 to the beads and incubate for 30 min on a rotating wheel at room temperature Resuspend the beads pellet and transfer it into a new 200 ul tube Briefly spin the tubes and place them into the Diagenode magnetic rack Transfer the supernatant to a new tube and add 4 ul of Elution buffer iE2 Also add 97 5 pl buffer iE1 and 4 ul of buffer iE2 to the 2 5 ul input sample Incubate for 4 hours or overnight at 65 C Purify the DNA using the IPure kit Add 2 ul of carrier to each IP and input sample Add 100 ul of 10096 isopropanol to each IP and input sample ATTENTION Following the addition of isopropanol the solution may become cloudy This is not detrimental to your experiment and will not influence the quality or quantity of your purified DNA Resuspend the IPure beads v2 and transfer 10 ul to each IP and inp
19. body used for the IP The protocol below uses 30 ul of beads The binding capacity of this amount is approximately 10 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 seq 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 1096 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 4 000 000 HeLa cells is approximately 20 ng www
20. butors php E Q Tel 32 4 364 20 50 Fax 1 862 209 4681 For rest of the world please contact Fax 32 4 364 20 51 orders na diagenode com Diagenode sa orders diagenode com info na diagenode com www diagenode com info diagenode com 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 iDealChlPseqTF V2_11_15
21. ccording to Table 3 If required NaBu 20 mM final concentration or other inhibitors can also be added Add 70 ul of ChIP reaction mix to the tubes containing the beads In table 3 x is the amount of antibody needed If you will use different amounts of antibody e g when performing a titration curve we recommend to add the water separately Table 3 ChIP reaction mix N of IP s 5 BSA pl 200x Protease inhibitor cocktail yl 5x buffer iC1b pl ChIP seq grade water pl Antibody ul 1 6 1 8 20 42 2 x x 2 12 3 3 40 84 4 x x 4 24 7 2 80 168 8 x x 6 36 10 8 120 253 2 x x 8 48 14 4 160 337 6 x Xx 21 Add the antibody to the reaction mix Use 1 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 pl of the CTCF positive control antibody www diagenode com diagenctie PAGE 14 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL 22 Incubate the tubes for 2 4 hours at 4 C under constant rotation at 40 rpm on a rotating wheel 23 Briefly spin the tubes and add 250 ul of sheared chromatin Put 2 5 ul of the sheared chromatin aside to be used as an input the next day Incubate the tubes overnight at 4 C under constant rotation at 40 rpm on a rotating wheel 24 The next morning after the overnight incubation briefly spin the tubes and place them in the magnetic rack Wait for one minute and remove the
22. diagenctie Innovating Epigenetic Solutions iDeal ChlP seq kit for Transcription Factors Cat No C01010055 24 rxns C01010170 100 rxns Version 2111 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 PAGE 3 Content Introductio SE ae ee nc ts ene es arte ve Bcc eh eg pags ee ee see ee 4 Kit Method Overview amp Time Table mener nnn n enne nns 5 Kit Materials oscuro rere ato ER b o ertet eben p E end tere E neca races dis Ec ee 5 Required Materials Not provided ss ss assure eects 6 Remarks before starting neces 7 Short Protocol 5e eter trt i eser D deeckcdter uci sees epit ee RUE C eer cupi irae Mon Feds di du puedes 9 Detailed Protocol ctr pce eR CD CR REN ER EROR ZEE ELUNGRAQR CUERO e DU ONE NON ES renen 12 STEP 1 Cell collection and DNA protein cross linking ssssssssssssR ees 12 STEP 2 Cell lysis and chromatin Shearg e
23. e 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 info ddiagenode 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 www diagenode com diagenctie diagenctie Innovating Epigenetic Solutions DIAGENODE S A DIAGENODE INC FOR A COMPLETE LISTING OF BELGIUM EUROPE USA NORTH AMERICA DIAGENODE S INTERNATIONAL LIEGE SCIENCE PARK 400 Morris Avenue Suite 101 DISTRIBUTORS VISIT DIAGENODE Rue Bois Saint Jean 3 Denville NJ 07834 http www diagenode com company HEADQUARTERS 4102 Seraing Belgium Tel 1 862 209 4680 distri
24. ely This equation assumes that the PCR is 10096 efficient amplification efficiency 2 For accurate results the real amplification efficiency if known should be used For the positive control antibody CTCF the recovery of the positive control target H19 imprinting control region is expected to be approximately 596 although this will depend on the cell type used The recovery of the negative control target Myoglobin exon 2 locus should be below 0 596 6 Figure 1 ChIP was performed on human HeLa cells using m H19 imprinting the control antibodies from the iDeal ChlP seq kit for control region Transcription Factors Sheared chromatin from 4 million m Myoglobin cells 0 5 ul of the positive control antibody and 1 ul of the exon 2 negative IgG control were used per IP Quantitative PCR 4 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 qPCR analysis of input Co CTCF IgG Innovating Epigenetic Solutions PAGE 9 Short protocol for experienced users STEP 1 Cell collection and DNA protein cross linking Note When studying inducible transcription factors or cofactors it is recommended to perform the fixation using the ChIP cross link Gold C01019027 in addition to the f
25. ents 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 information 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 nontransferable 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 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 sol
26. esse an nee mme em ener nnne 12 STEP 3 Magnetic immuno precipitato core ertet ern Fer pepe teu dodi as Wer qi o pedido drugs 13 STEP 4 Elution decross linking and DNA isolation ssssssssssssssss e ees 14 STEP 5 Quantitative PCR analysis ssssssssssssssss eene e n enne esses ne e e nens 15 ChIP sequsncirng eroe eB EU LU RM ML Me 16 ChIP seq data analysis recommendations sss 17 Additional Protocols n re rtm ete E tete vn Pe et eese Dog pte da get etie nuts 20 Troubleshooting guide nnn eee ees 22 www diagenode com diagenctie PAGE 4 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Introduction Association between proteins and DNA is a major mechanism in many vital cellular functions such as gene transcription and epigenetic silencing It is crucial to understand these interactions and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation Chromatin immunoprecipitation ChIP is a technique to analyze the association of proteins with specific genomic regions in intact cells ChIP can be used to study changes in epigenetic signatures chromatin remodelling and transcription regulator recruitment to specific genomic sites The different steps of the ChIP assay are cell fixation cross linking chromatin shearing immunoprecipitation reverse cross linking followed by DNA purification and analysis of the immunoprecipitated DNA In ChIP
27. he IP and input samples with the Wash buffer 2 as follows Briefly spin the tubes place into the DiaMag1 5 wait 1 min and discard the buffer Add 100 ul Wash buffer 2 per tube Close the tubes and vortex well until the beads pellet is completely broken In order to avoid the beads pellet to be too difficult to break down do not let the beads for too long on the magnet incubate for 30 seconds at room temperature on a rotating wheel 40 rpm e Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning in the DiaMag rack NOTE This Elution buffer buffer C is suitable for direct qPCR analysis whole genome amplification chip hybridization and Next Generation sequencing 37 a Briefly spin the tubes and place them into the DiaMag02 wait 1 min and discard the buffer Spin the tubes again and place them on the DiaMag02 magnetic rack Discard the remaing Wash buffer 2 if necessary Resuspend the beads pellet with 25 ul of buffer C Incubate at room temperature for 15 minutes on a rotating wheel 40 rpm b Spin the tubes and place them into the DiaMag1 5 wait 1 min and transfer the supernatants into a new labelled 1 5 ml tube and discard the beads c Place the DNA on ice and proceed to any desired downstream applications or store it at 20 C or 80 C until further use 40 Take 5 ul 10 of IP d DNA and determine the concentration with Quant IT dsDNA HS assay kit using the Qubit system or a similar method
28. hyde concentration e g incubate for 10 20 minutes at room temperature with 1 96 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 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 196 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 sheared Efficient fixation of a protein to chromatin in vivo is a crucial step
29. imization and sheared chromatin analysis Before starting the ChIP the chromatin should be sheared 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 anti
30. ion 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 transcription 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
31. 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 16 Determine the total nr of IP s in the experiment Please note that we recommend to include one negative control in each experiment IP with the IgG negative control Take the required amount of DiaMag Protein A coated magnetic beads 30 ul IP Prepare 4 ml of 1x ChIP buffer iC1b by adding 3 2 ml ChIP seq grade water to 0 8 ml 5x ChIP buffer iC1b Add 80 ul of 5 BSA Keep the diluted ChIP buffer iC1 on ice 17 Wash the beads 3 times with 1 ml of ice cold 1x ChIP buffer iC1b To wash the beads add 1x ChIP buffer iC1 resuspend the beads by pipetting up and down several times and incubate at 4 C with gentle shaking for 5 Spin the tubes and place them in the 1 5 ml Diagenode magnetic rack Cat No B04000003 Wait for one minute to allow the beads to be captured by the magnet and remove the supernatant Repeat this 2 times Alternatively you can centrifuge the tubes for 5 minutes at 1 300 rpm discard the supernatant and keep the bead pellet 18 After the last wash resuspend the beads in the original volume 1x ChIP Buffer iC1b 19 Take the required number of tube and add 30 ul of the washed beads to each tube 20 Prepare the ChIP reaction mix a
32. ntrolled kits reagents and equipment is a perfect starting point that will lead to your success Innovating Epigenetic Solutions Kit method overview amp time table Table 1 iDeal ChIP protocol overview Step Time needed Day 1 Cell collection and DNA protein cross linking for cultured cells 1 to 2 hours 1 2 Cell lysis and chromatin shearing for cultured cells 1 to 2 hours 1 3 Magnetic immunoprecipitation overnight 1 2 4 Elution decross linking and DNA purification 5 hours 2 5 Quantitative PCR and data analysis prior to Library preparation and Next Generation Sequencing 2 to 3 hours 3 Kit materials The iDeal ChIP seq kit for Transcription Factors x100 contains reagents to perform 17 different chromatin preparations 100 Chromatin Immunoprecipitations and DNA Purificationfrom cell collection to IP d DNA The iDeal ChIP seq kit for Transcription Factors x24 contains reagents to perform 4 different chromatin preparations 24 Chromatin Immunoprecipitations and DNA Purificationfrom cell collection to IP d DNA The kit content is described in Table 2 Upon receipt store the components at the temperatures indicated in Table 2 Table 2 Components supplied with the iDeal ChIP seq kit Description Quantity x24 Quantity x100 Storage Protease inhibitor cocktail 95 ul 385 ul 20 C 5 BSA DNA free 940 ul 3
33. oose either protein A or protein G coated beads Species Immunglobulli Isotype Protein A Protein G Human g61 gG2 963 a Antibody in IP gG4 p qu gGM Use anti Human IgM gGF gGA Mouse g61 re my antibodies going to bind the gG2a protein A or protein G gG2b b n 963 gGM Use anti Mouse IgM Rat g61 5 gG2a amp gG2b E m gG2c Chicken ALL Isotypes Cow All Isotypes Goat All Isotypes Guinea Pig All Isotypes Hamster All Isotypes Horse All Isotypes Pig All Isotypes Rabbit All Isotypes Sheep All Isotypes Freezing Avoid multiple freeze thawing Snap freeze and thaw on ice e g fixed cell pellets and sheared chromatin Innovating Epigenetic Solutions PAGE 23 FOR RESEARCH USE ONLY Not intended for any animal or human therapeutic or diagnostic use 2014 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 electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from Diagenode SA hereinafter Diagenode 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 developm
34. ormaldehyde fixation 1 Dilute formaldehyde in Fixation buffer to a final concentration of 11 2 Add 1 10 volume of diluted formaldehyde directly to the cell culture medium 3 Incubate at room temperature for 10 to 20 minutes with gentle shaking 4 Stop the fixation by adding 1 10 volume of glycine STEP 2 Cell lysis and chromatin shearing EEE For adherent cells 25 million 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 Rinse the flask with an additional 20 ml of buffer iL1b 8a Incubate at 4 C for 20 minutes For suspension cells 25 million cells 5b Collect the cells by centrifugation at 1 600 rpm and 4 C for 5 minutes 6b Wash the cells once with 20 ml of PBS Keep everything at 4 C from now on 7b Resuspend the cells in 25 ml of cold lysis buffer iL1b 8b Incubate at 4 C for 20 minutes 9 Discard the supernatant and resuspend the cells in 15 ml lysis buffer iL2 Incubate at 4 C for 10 minutes with gentle mixing 10 Centrifuge at 1 600 rpm and 4 C for 5 minutes 11 Add 1 200 volume protease inhibitor cocktail PIC to shearing buffer iS1b 12 Discard the supernatant and resuspend the cells in shearing buffer iS1b PIC to a final concentration of 15 million cells ml Resuspend by pipetting up and down 13 Shear chromatin by sonication using the Bior
35. quencing 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 E custsupport diagenode com Contact for North and South America ASK THE custsupport na diagenode com EXPERTS ChIP seq 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 built in aligners with default settings worked very well for our ChIP seq experiments e g ELAND for Ilumina 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 you
36. r 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 Innovating Epigenetic Solutions PAGE 17 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 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 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 calculat
37. ruptor Pico Innovating Epigenetic Solutions PAGE 13 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 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 STEP 3 Magnetic immunoprecipitation The immunoprecipitation step can also be performed using the semi automated ChiPettor System If doing so please refer to the corresponding protocol delivered an with this product Alternatively the protocol can also be downloaded from the website ee e www diagenode com 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
38. tibody with saturating amounts of llel its epitope specific peptide for about 30 minutes at room temperature before use in the parar P incubation mix Use the blocked antibody as a negative control in parallel with the unblocked antibody H ti tral f multiple antibodies of the same species are to be used with the same chromatin ON AED AE DSA SENSE SE preparation then a single negative ChIP control is sufficient for all of the antibodies necessary used Why is my antibody not working in Antibody antigen recognition can be significantly affected by the cross linking step ChIP 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 Which antibody should ChIP i Ru MELLE LER 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 Be aware of the possible cross reactivity of antibodies Verify by Western blot analysis How do choose an antibody for ChIP 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 different types of immunoglobulins to protein A or G Thererfore in function of the antibody used for your ChIP it is recommended to ch
39. 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 www diagenode com diagenctie PAGE 18 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL also be used e g the FastQC by Babraham Institute http www bioinformatics bbsrc ac uk projects fastqc 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 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 Tsi p M oM g m ett m mm mu B n a ie A TET on a o 14 p OVON COPSTA LARCH CIS Figure 5 Various stages of bioinformatics data analysis
40. tus Ghr matin Critical points for shearing 1 Not to start with a too high concentration of cells 15 x 10e6 cells ml or less is ok optimization 2 Keep samples cold 4 C shearing Shear the samples of chromatin using Maintain temperature of the samples close to 0 C The chromatin shearing needs to be the Bioruptor Pico from Diagenode optimized for each cell type A troubleshooting guide for Bioruptor chromatin shearing is available at Diagenode Purify the DNA from the sheared Extract total DNA from an aliquot of sheared chromatin and run on 196 agarose gel chromatin as described in the kit stain with EtBr In order to analyse the sheared chromatin on gel take DNA purified protocol to analyse the shearing 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 Sheared or more can for sure be visualized on a gel chromatin Do not load too much DNA on a gel Loading of large quantities of DNA on agarose gel can lead to poor quality pictures analysis which do not reflect the real DNA fragmentation The DNA amount to load depends on he size of the well and on the gel size Agarose concentration Do not use more than 1 1 5 agarose gel and run slowly Volt cm and time depend on he gel size Running buffer concentration 1x TAE or TBE is preferred to 0 5x
41. uptor An initial time course experiment is recommended For Bioruptor Standard or Plus use High power setting for 10 30 cycles 30 seconds ON 30 seconds OFF Stop he system after each run of 10 cycles vortex and spin down sample For Bioruptor Pico sonicate samples for 8 10 cycles 30 seconds ON 30 seconds OFF Vortexing is not required between runs 14 Centrifuge at 13 000 rpm 16 000 x g for 10 minutes and collect the supernatant which contains the sheared chromatin www diagenode com diagenctie PAGE 10 DIAGENODE IDEAL CHIP SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL STEP 3 Magnetic immunoprecipitation The immunoprecipitation step can also be performed using the semi automated ChiPettor System If doing so please refer to the corresponding protocol delivered with this product Alternatively the protocol can also be downloaded from the website www diagenode com 15 16 17 18 19 20 21 22 23 24 25 Prepare 4 ml of 1x ChIP buffer iC1b Add 80 ul of 5 BSA Take the required amount of magnetic beads 30 pl ChIP and was them 3 times with 1 ml 1x ChIP buffer iC1b BSA Resuspend the beads in the original volume of 1x ChIP buffer iC1b BSA Prepare the following ChIP reaction mix 6 ul of 5 BSA 1 8 ul of 200x PIC 20 ul of 5x ChIP buffer iC1b 42 2 uL H20 30 ul beads Add the required amount of antibody Incubate for 2 4 hours or overnight at 4 C on a rot
42. ut sample Vortex well and incubate 10 minutes at room temperature on a rotating wheel 40 rpm Prepare the Wash buffer 1 containing 50 isopropanol Wash buffer 1 24 rxns 100 rxns Wash buffer 1 w o isopropanol 2ml 8ml Isopropanol 100 2ml 8ml Total volume 4ml 16 ml Innovating Epigenetic Solutions PAGE 11 35 Briefly spin the tubes place into the DiaMag0 2 magnetic rack wait 1 min and discard the buffer Add 100 ul Wash buffer 1 in each tube Close the tubes incubate for 30 seconds at room temperature on a rotating wheel 40 rpm 36 Prepare the Wash buffer 2 containing 5096 isopropanol as follows Wash buffer 2 24 rxns 100 rxns Wash buffer 2 w o isopropanol 2ml 8ml Isopropanol 100 2ml 8ml Total volume 4ml 16 ml 38 Briefly spin the tubes place into the DiaMag0 2 magnetic rack wait 1 min and discard the buffer Keep the captured beads and add 100 ul Wash buffer 2 per tube Close the tubes resuspend the beads and incubate for 5 min at room temperature on a rotating wheel 40 rpm 39 Briefly spin the tubes and place them into the DiaMag0 2 or DiaMag1 5 wait 1 minute and discard the buffer Keep the captured beads and add per tube 25 ul buffer C alternatively a higher volume can be used for the elution if necessary Close the tubes invert the 8 tube strip to resuspend the beads and incubate for 15 minutes at room temperature on a rotating wheel 40 rpm
43. yde 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 C01010050 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 1 596 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 graph In this example the optimal shearing condition corresponds to 3 rounds of 10 cycles 30 sec ON 30 sec OFF Innovating Epigenetic Solutions Troubleshooting guide Critical steps Troubles solutions and comments Cross linking Cross linking is too weak Cross linking is too strong Make sure you perform the fixation step for the correct period of time at the right temperature and with the correct formalde
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