EpiNext™ ChIP-Seq High
Contents
1. EpiNext ChIP Seq High Sensitivity Kit Base Catalog P 2030 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext ChIP Seq High Sensitivity Kit is designed to selectively enrich a chromatin fraction containing specific DNA sequences from various species particularly mammals and to prepare a ChIP Seq library for next generation sequencing using Illumina platforms such as Illumina Genome Analyzer II HiSeq and MiSeq systems The optimized protocol and components of the kit allow capture of low abundance protein DNA complexes with minimized non specific background levels and the ability to construct both non barcoded singleplexed and barcoded multiplexed ChIP Seq libraries quickly with reduced bias Input Amount of Tissue Cells In general the amount of cells and tissues for each reaction can be 1 x 10 to 1 x 10 and 5 mg to 50 mg respectively For optimal preparation the input amount should be 4 to 5 x 10 cells or 20 to 30 mg tissues so that the amount of DNA enriched from the ChIP reaction can range from at least 1 ng to 100 ng Starting Materials Starting materials can include various tissue or cell samples such as culture cells from a flask or plate fresh and frozen tissues etc Antibodies Antibodies should be ChIP grade in order to recognize fixed and native proteins that are bound to DNA or other proteins If you are using antibodies which have not been validated for ChIP then appropriate control antibodi2. Grow Cells treated or untreated to 80 90 confluence on a 6 well plate or 100 mm dish the number of cultured MDA 231 cancer cells on an 80 90 confluent plate is listed in the table below as a reference then trypsinize and collect them into a 15 ml conical tube Count the cells ina hemocytometer 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 Container Cell Number x 10 96 well plate 0 3 0 6 well 24 well plate 1 3 well 12 well plate 3 6 well 6 well plate 5 10 well 60 mm dish 20 30 100 mm dish 50 100 150 mm dish 150 180 b Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant c Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3 1 i after Step 3 1 c d Add 9 ml fresh cell culture medium containing formaldehyde to a final concentration of 1 i e add 270 ul of 37 formaldehyde to 10 ml of cell culture medium to cells e Incubate at room temperature 20 25 C for 10 min on a rocking platform 50 100 rpm f Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution g Mix and centrifuge at 1000 rpm for 5 min h Remove medium and wash cells once with
3. Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads i Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear j Transfer 20 ul to a new 0 2 ml PCR tube Quality of the prepared library can be assessed using an Agilent Bioanalyzer or other comparable methods Library fragments should have the correct size distribution e g 300 400 bps at peak size without adaptors or adaptor dimers To check the size distribution dilute library 5 fold with water and apply it to an Agilent high sensitivity chip If there is presence of lt 150 bp adaptor dimers or of larger fragments than expected they should be removed To remove fragments below 150 bps or above 500 bps use 0 8X MQ Binding Beads according to sub steps a through of Step 11 1 Size Selection of Ligated DNA Store the prepared library at 20 C until ready to use for sequencing TROUBLESHOOTING For ChIP Reaction Problem Possible Cause Suggestion Little or no PCR Poor chromatin quality due to The optimal amount of chromatin per products insufficient amount of cells or ChIP reaction should be 2 4 ug about 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 16 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for resear
4. beads for 10 minutes while the tube is on the magnetic stand Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 11 ul to a new 0 2 ml PCR tube for PCR amplification 12 Library Amplification a Prepare the PCR reactions Thaw all reaction components including master mix DNA RNA free water primer solution and DNA template Mix well by vortexing briefly Keep components on ice while in use and return to 20 C immediately following use Add components into each PCR tube well according to the following table Component Size ul HiFi Master Mix 2X 12 5 ul Primer U 1 ul Primer 1 ul Adaptor Ligated DNA 10 5 ul Total Volume 25 ul Important Note Use of Primer I included in the kit will generate a singleplexed library For multiplexed library preparation replace Primer I with one of the12 different barcodes indexes contained in the EpiNext NGS Barcode Index Set 12 Cat No P 1060 You can also add user defined barcodes Illumina compatible instead of Primer I b Program the PCR reactions Place the reaction plate in the instrument and set the PCR conditions as follow 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 15 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www
5. epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 Cycle Step Temp Time Cycle Activation 98 C 30 sec 1 98 C 20 sec Cycling 55 C 20 sec Variable 72 C 20 sec Final Extension 72 C 2 min 1 PCR cycles may vary depending on the input DNA amount In general use 8 PCR cycles for 100 ng 10 cycles for 50 ng 13 cycles for 5 ng 15 cycles for 1 ng and 18 cycles for 0 2 ng DNA input Further optimization of PCR cycle number may be required 13 Clean Up of Amplified Library DNA a Resuspend MQ Binding Beads by vortex b Add 25 ul of resuspended beads to the PCR tube of amplification reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Cc Incubate for 5 minutes at room temperature to allow DNA to bind to beads d Put the PCR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA e Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol f Repeat Step e two times for total of three washes g Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand h Resuspend the beads in 22 ul
6. m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 m Non immune IgG RNA polymerase Il antibody Relative Enrichment Fold 0 2 000 4 000 20 000 100 000 500 000 Cell Number Fig 2 High sensitive ChIP The sheared chromatin isolated from different number of MBD 231 cells was used fo ChIP qPCR analysis of RNA polymerase II enrichment in GAPDH promoters FU 60 50 40 30 50 300 500 700 1000 3000 10330 bp Fig3 Size distribution of library fragments Ten nanograms of DNA was ChiPed by RNA polymerase II enrichment and used for DNA library preparation ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment ChIP Reaction 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 1 Preparation of Working Buffers and Solutions a Prepare Working Lysis Buffer by adding 6 ul of Protease Inhibitor Cocktail to every 10 ml of LB Lysis Buffer Prepare Working CB ChIP buffer by adding 1 ul of Protease Inhibitor Cocktail to every 1 ml of CB ChIP buffer 2 Antibody Binding to Strip Wells a Predetermine the number
7. m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only P 2030 Remove medium and wash cells once with 10 ml of ice cold PBS by centrifuging at 1000 rpm for 5 min Add Working Lysis Buffer to re suspend the cell pellet 200 1l 1x10 cells and incubate on ice for 10 min Vortex vigorously for 10 sec and centrifuge at 3000 rpm for 5 min Then go to Step 4a For Tissues Put the tissue sample into a 60 or 100 mm plate Remove unwanted tissue such as fat and necrotic material from the sample Weigh the sample and cut the sample into small pieces 1 2 mm with a scalpel or scissors Note For tissues that are not cross linked go directly to Step 3 3 j after Step 3 3 b Transfer tissue pieces to a 15 ml conical tube Prepare cross link solution by adding formaldehyde to cell culture medium to a final concentration of 1 e g add 270 ul of 37 formaldehyde to 10 ml of culture medium Add 1 ml of cross link solution for every 50 mg tissues Incubate at room temperature for 15 20 min on a rocking platform Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution Mix and centrifuge at 800 rpm for 5 min Discard the supernatant Wash cells with 10 ml of ice cold PBS once by centrifugation at 800 rpm for 5 min Discard the supernatant Transfer tissue pieces to a Dounce homogenizer Add 0 5 ml Working Lysis Buffer for every
8. of strip wells required for your experiment Carefully remove unneeded strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C Setup the antibody binding reactions by adding the reagents to each well according to the following chart Reagents Sample Positive Control Negative Control AB Antibody Buffer 50 80 ul 50 80 ul 50 80 ul Your Antibodies 0 5 2 ul 0 0 Anti RNA Polymerase II 0 0 8 ul 0 Non Immune IgG 0 0 0 8 ul Note The final amount of each component should be a antibodies of interest 0 8 ug well b RNA Polymerase II 0 8 ug well and c non immune IgG 0 8 ug well The amount of the positive control RNA polymerase II and negative control Non lmmune IgG are sufficient for matched use with samples if two antibodies are used for each sample or one antibody is used for two of the same samples If using one antibody of interest for each sample with matched use of the positive and negative control extra RNA polymerase II non immune IgG and 8 well strips are required and can be separately obtained from Epigentek Seal the wells with Adhesive Covering Film Strips and incubate the wells at room temperature for 60 90 min on an orbital shaker 100 rpm Meanwhile perform the steps from Section 3 Cell Collection and Cross Linking to Section 5 Chromatin Shearing 3 Cell Collection and Cross Linking 3 1 For Monolayer or Adherent Cells a
9. 0 000 cells with a range from 50 000 to 1 000 000 cells Broad range of cell tissue samples can be used including samples with limited amount e Fast and streamlined procedure The procedure from cell tissues to library DNA is less than 7 hours No clean up is required between each step from ChiPed DNA to size selection and all reactions take place in the same tube thereby saving time and preventing handling errors or loss of valuable samples Gel free size selection further reduces the preparation time e Highly convenient for use The kit contains all required components for each step of ChIP Seq which are sufficient for both ChIP and ChiPed DNA library preparation thereby allowing the ChIP Seq to be the most convenient with reliable and consistent results e Minimized bias Ultra HiFi amplification and optional PCR free step allow achievement of reproducibly high yields of DNA library with minimal sequence bias and low error rates PRINCIPLE amp PROCEDURE 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 07 29 P 2030 The EpiNext ChIP Seq High Sensitivity Kit contains all necessary reagents required for carrying out a successful ChIP Seq starting from mammalian cells or tissues In the ChIP reaction chromatin is isolated from ce
10. 0 mg ml 30 ul 60 ul 4 RNase A 10 mg ml 15 ul 30 ul 20 C GAPDH Primer Forward 20 uM 5 ul 10 ul 4 GAPDH Primer Reverse 20 uM 5 ul 10 ul 4 8 Well Assay Strips With 1 Frame 2 4 47 8 Well Strip Caps 2 4 RT Adhesive Covering Film Strip 4 8 RT F Spin Column 15 30 RT F Collection Tube 15 30 RT 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2014 07 29 P 2030 For Library Preparation Component 12 reactions 24 reactions Storage Cat P 2030 12 Cat P 2030 24 Upon Receipt 10X End Polishing Buffer 30 ul 60 ul 20 C End Polishing Enzyme Mix 13 ul 26 ul 20 C End Polishing Enhancer 13 ul 26 ul 20 C 2X Ligation Buffer 250 ul 500 ul 20 C T4 DNA Ligase 15 ul 30 ul 20 C Adaptors 50 uM 15 ul 30 ul 20 C MQ Binding Beads 1 6 ml 3 2 ml 4 C 2X HiFi PCR Master Mix 160 ul 320 ul 20 C Primer U 10 uM 15 ul 30 ul 20 C Primer 10 uM 15 ul 30 ul 20 C Elution Buffer 1000 ul 2000 ul 20 C User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt Store the followi
11. 01 P 2023 ChromaFlash Chromatin Extraction Kit ChromaFlash Chromatin lsolation Shearing Kit DNA Isolation and Cleanup P 1003 P 1004 P 1006 P 1007 P 1009 P 1017 P 1018 FitAmp General Tissue Section DNA Isolation Kit FitAmp Plasma Serum DNA Isolation Kit DNA Concentrator Kit FitAmp Gel DNA Isolation Kit FitAmp Paraffin Tissue Section DNA Isolation Kit FitAmp Urine DNA Isolation Kit FitAmp Blood and Cultured Cell DNA Extraction Kit Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 DNA Enrichment Reaction P 1015 P 1038 P 1052 P 2002 P 2003 P 2014 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Methylamp Methylated DNA Capture Kit EpiQuik Hydroxymethylated DNA Immunoprecipitation nMeDIP Kit EpiQuik MeDIP Ultra Kit EpiQuik Chromatin Immunoprecipitation Kit EpiQuik Tissue Chromatin Immunoprecipitation Tissue Kit EpiQuik Plant ChIP Kit Page 19 Printed 2014 07 29 P 2030 P 2025 ChromaFlash One Step ChIP Kit P 2026 ChromaFlash One Step Magnetic ChIP kit P 2027 ChromaFlash ChIP Ultra Kit PCR Analysis P 1029 EpiQuik Quantitative PCR Kit DNA Library Prep P 1051 EpiNext DNA Library Preparation Kit Illumina P 1053 EpiNext High Se
12. 10 ml of ice cold PBS by centrifuging at 1000 rpm for 5 min i Add Working Lysis Buffer to re suspend the cell pellet 200 ul 1x10 cells and incubate on ice for 10 min Note If the total solution volume is less than 1 5 ml transfer the solution to a 1 5 ml microtube j Vortex vigorously for 10 sec then centrifuge at 3000 rpm for 5 min Go to Step 4a 3 2 For Suspension Cells a Collect cells treated or untreated into a 15 ml conical tube 2 x10 to 5x10 cells are required for each ChIP reaction Count cells in a hemocytometer b Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant c Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3 2 1 after Step 3 2 c d Add 9 ml fresh cell culture medium containing formaldehyde to a final concentration of 1 i e add 270 ul of 37 formaldehyde to 10 ml of cell culture medium to cells e Incubate at room temperature 20 25 C for 10 min on a rocking platform 50 100 rpm f Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution g Mix and centrifuge at 1000 rpm for 5 min 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 07 29 P 2030 Tel 1 877 374 4368
13. 50 mg of tissues Disaggregate tissue pieces by 20 40 strokes Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 min at 4 C If total mixture volume is less than 2 ml transfer mixture to a 2 ml vial and centrifuge at 5000 rpm for 5 min at 4 C Then go to Step 4a 4 Cell Lysis and Chromatin Extraction a Carefully remove supernatant b Add CB ChIP Buffer to re suspend the chromatin pellet 100 l 1x10 cells or 50 mg tissue 500 ul maximum for each vial c Transfer the chromatin lysate to a 1 5 ml vial and incubate on ice for 10 min and vortex occasionally 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 10 Printed 2014 07 29 5 Chromatin Shearing a Resuspend the chromatin lysate by vortexing b Shear chromatin with one of the following methods Waterbath Sonication Epigentek EpiSonic 1100 Epigentek Cat No EQC 1100 Use 50 ul of chromatin lysate per 0 2 ml tube or per PCR plate well Shear 20 cycles under cooling condition 30 seconds ON 30 seconds OFF each at 170 190 watts For more detailed information of use please see the Chromatin Shearing Protocol for EpiSonic 1100 If using other waterbath sonicators please follow the supplier s instruction Probe based Sonication Use 300 ul of chromatin lysate per 1 5 ml microcentrifuge tube As an example sonication can be carried out with a microtip attached to a Branson 450 sonifier set to 25 power output Sonica
14. and discard the ethanol h Repeat Step g one time for total of two washes i Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand j Resuspend the beads in 12 ul Elution Buffer and incubate at room temperature for 2 minutes to release the DNA from the beads 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 14 Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 k Capture the beads by placing the tube in the magnetic stand for 4 minutes or until the solution is completely clear Transfer 11 ul to a new 0 2 ml PCR tube for PCR amplification 11 2 Clean up of Ligated DNA Optional Resuspend MQ Binding Beads by vortex Add 34 ul of resuspended beads to the PCR tube of ligation reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 5 minutes at room temperature to allow DNA to bind to beads Put the PCR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 90 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step e two times for total of three washes Open the PCR tube cap and air dry
15. and then removing it 8 Reversal of Cross Links Release and Purification of DNA a Prepare RNase A solution by adding 1 ul of RNase A to 400 ul of DRB b Add 40 ul of DRB RNase A to each well and then cover with a strip cap c Incubate the wells at 42 C for 30 min d Add 2 ul of Proteinase K to each well and re cap the wells e Incubate the wells at 60 C for 30 min Quickly transfer the DNA solution from each well to 0 2 ml strip PCR tubes Cap the PCR tubes 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 12 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 g Incubate the PCR tubes containing DNA solution at 95 C for 15 min in a thermolcycler h Place the PCR tubes at room temperature If liquid is collected on the inside of the caps briefly spin the liquid down to the bottom i Place a spin column into a 2 ml collection tube Add 200 ul of DBS DNA Binding Solution to the samples and transfer mixed solution to the column Centrifuge at 12 000 rpm for 30 seconds j Add 200 ul of 90 ethanol to the column centrifuge at 12 000 rpm for 30 seconds Remove the column from the collection tube and discard the flowthrough k Replace column to the collection tube Add 200 ul of 90 ethanol to the column and centrifuge at 12 000 rpm for 30 seconds Remove th
16. ant role in identifying genome wide protein DNA interaction However these methods still have several drawbacks 1 large amounts of cell tissues are needed for obtaining a sufficient yield of library DNA therefore these methods cannot be used for biological samples such as tumor biopsy and embryonic tissues whose amounts are limited 2 the background levels of ChlPed DNA are high and 3 the procedures are time consuming gt 3 days and inconvenient To address this issue Epigentek developed the EpiNext ChIP Seq High Sensitivity Kit by combining its microplate based ultra ChIP and high sensitive DNA library construction technologies This kit has the following features e Optimized buffers and protocol allow minimal ChIP background by overcoming the weaknesses that cause non specific enrichment thereby increasing sensitivity and specificity of the ChIP reaction e Increased antibody selectivity and capture efficiency through the use of unique chimeric proteins containing the maximum number of IgG binding domains coated on the strip wells This allows strong binding of any IgG subtype antibodies within a wide pH range regardless of monoclonal or polyclonal form e Highly efficient enrichment of targeted DNA Enrichment ratio of positive to negative control gt 500 e High sensitivity and flexibility Can be used for both non barcoded singleplexed and barcoded multiplexed DNA library preparation The input cell number can be as few as 5
17. ch use only P 2030 generated from both sample and positive control wells insufficient or over cross linking 2 4 x 10 cells Appropriate chromatin cross linking is also required Insufficient or over crosslinking will cause DNA loss or increased background During the cross linking step of chromatin preparation ensure that the cross linking time is within 10 15 min the final concentration of formaldehyde is 1 and the quench solution is 0 125 M glycine Poor enrichment with antibody some antibodies used in ChIP might not efficiently recognize fixed protein Increase the antibody amount and use ChIP grade antibodies validated for use in ChIP Inappropriate DNA fragmenting condition If chromatin is from specific cell tissue types or is differently fixed the shearing conditions should be optimized to allow DNA fragment size to be between 100 700 bp Incorrect temperature and or insufficient time during DNA release Ensure the incubation times and temperatures described in the protocol are followed correctly Improper PCR conditions including improper PCR programming PCR reaction solutions and or primers Ensure the PCR is properly programmed If using a homebrew PCR reaction solution check if each component is correctly mixed If using a PCR commercial kit check if it is suitable for your PCR Confirm species specificity of primers Primers should be designed to cover a sh
18. e column and discard the flowthrough Replace column to the collection tube and wash the column again with 200 ul of 90 ethanol at 12 000 rpm for 1 min m Place the column in a new 1 5 ml vial Add 11 ul of DEB DNA Elution Buffer directly to the filter in the column and centrifuge at 12 000 rpm for 30 seconds to elute purified DNA Purified DNA is now ready for ChlPed DNA library preparation after verifying the quality and amount of the ChliPed DNA by qPCR or a suitable fluorescence method Note For real time PCR analysis we recommend the use of 1ul of eluted DNA in a 20 ul PCR reaction If input DNA will be used it should be diluted 10 fold before adding to PCR reaction Control primers 110 bp for human cells included in the kit can be used as a positive control In general the amplification difference between normal IgG control and positive control may vary from 3 to 8 cycles depending on experimental conditions Optimally 10 ng of ChiPed DNA are required for ChIP DNA library construction This amount can be easily generated for high abundance targets from a single ChIP reaction well However this may be difficult for low abundance target enrichment We recommend pooling the DNA solution from several low abundance ChIP reaction wells to gain 10 ng or more of DNA ChiPed DNA Library Preparation 9 DNA End Polishing a Prepare end repair reaction in a 0 2 ml PCR tube according to Table 1 Table 1 End Polishing React
19. es such as RNA Polymerase II Cat A 2032 should be used to demonstrate that the antibody and prepared chromatin are suitable for ChIP Internal Controls Both negative and positive ChIP controls are provided in this kit Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 07 29 P 2030 KIT CONTENTS For ChIP Reaction Component 12 reactions 24 reactions Storage P 2030 12 P 2030 24 Upon Receipt WB Wash Buffer 12 ml 25 ml 47 AB Antibody Buffer 1 ml 2ml 47 LB Lysis Buffer 7 ml 14 ml RT CB ChIP Buffer 6 ml 12 ml 4 DRB DNA Release Buffer 7 mi 14 ml RT DBS DNA Binding Solution 7 ml 14 ml RT BS Blocker Solution 1 ml 2 ml 47 DEB DNA Elution Buffer 0 5 ml 1 ml RT Enrichment Enhance 25 ul 50 ul 20 C Protease Inhibitor Cocktail PIC 15 ul 30 ul 4 Non Immune IgG 1 mg ml 5 ul 10 ul 47 Anti RNA Polymerase II 1 mg ml 5 ul 10 ul 47 Proteinase K 1
20. for 10 min 3 spin the solution down to the bottom 4 transfer supernatant to a new 0 2 ml PCR vial Use 30 40 ul for DNA fragment size analysis along with a DNA marker on a 1 2 agarose gel and 5 stain with ethidium bromide or other fluorescent dye for DNA and visualize it under ultraviolet light 6 Preparation of ChIP Reaction a Peel away the Adhesive Covering Film on the antibody binding wells from Step 2b carefully to avoid contamination between each well b Remove the antibody reaction solution and non immune IgG solution from each well and wash the wells one time with 150 ul of CB ChIP Buffer 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 c Setup the ChIP reactions by adding the reagents to the wells that are bound with antibodies sample and positive control wells or IgG negative control well according to the following chart Reagents Sample Positive Control Negative Control CB ChIP Buffer 50 80 ul 50 80 ul 50 80 ul Chromatin 10 40 ul 10 40 ul 10 40 ul Enrichment Enhancer 2 ul 2 ul 2 ul BS Blocker Solution 10 ul 10 ul 10 ul Note The final amount of chromatin should be 2 ug well 2 x 10 cells may yield 1 ug of chromatin Sonicated chromatin can be further diluted
21. ibrary Orbital shaker Magnetic stand 96 well PCR plate format Adjustable pipette and pipette tips 0 2 ml or 0 5 ml PCR vials 1 5 ml microcentrifuge tubes 15 ml conical tube Antibodies of interest Cells or tissues 100 ethanol Distilled water Cell culture medium 37 formaldehyde if cross linked 1 25 M glycine solution if cross linked Oo o o o Oo Oo oO o Oo o o O Oo o O oO GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiNext ChIP Seq High Sensitivity Kit is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions 1X PBS Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiNext ChIP Seq High Sensitivity Kit is for research use
22. including DNA End Polishing 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 18 Printed 2014 07 29 P 2030 Adaptor Ligation Size Selection and Amplification Improper storage of the kit Ensure that the kit has not exceeded the expiration date Standard shelf life when siored properly is 6 months from date of receipt presence of larger fragments than expected Unexpected peak size Improper ratio of MQ Binding Check if the correct volume of MQ of Agilent Beads to DNA volume in size Binding Beads is added to DNA Bioanalyzer trace selection solution accordingly Proper ratios presence of lt 150 bp should remove the fragments with adaptor dimmers or unexpected peak sizes Insufficient ligation Too much and too little input DNA may cause insufficient ligation which can shift peak size of the fragment population to be shorter or larger than expected Make sure that ligation reaction is properly processed with the proper amount of input DNA Over amplification of library PCR artifacts from over amplification of the library may cause the fragment population to shift higher than expected Make sure to use proper PCR cycles to avoid this problem RELATED PRODUCTS Chromatin Preparation P 20
23. ion ChiPed DNA 10 ul 10X End Polishing Buffer 1 5 ul End Polishing Enzyme Mix 1 ul End Polishing Enhancer 1 ul Distilled Water 1 5 ul Total Volume 15 pl b Mix and incubate for 20 min at 25 C and 20 min at 72 C in a thermocycler without heated lid 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 13 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030 Note The amount of fragmented DNA can be 0 2 100 ng with an optimal amount of 10 50 ng 10 Adaptor Ligation Prepare a reaction mix for adaptor ligation according to Table 2 Add the following reagents to a 0 2 ml PCR tube containing end repaired dA tailing end polished DNA from Step 9 Table 2 Adaptor Ligation End polished DNA from step 9 15 ul 2X Ligation Buffer 17 pl T4 DNA Ligase 1 ul Adaptors 1 ul Total volume 34 ul Mix and incubate for 15 min at 25 C in a thermocycler without heated lid Note 1 The pre annealed adapters included in the kit are suitable for both non barcoded singleplexed and barcoded multiplexed DNA library preparation and are fully compatible with Illumina platforms such as MiSeq or HiSeq sequencers 2 If using adaptors from other suppliers both single end and barcode adaptors make sure they are compatible with Illumina pla
24. ll tissues and the target protein DNA complex is immunoprecipitated using the antibody of interest Immunoprecipitated DNA is then cleaned released and eluted Included in the kit are a positive control antibody RNA polymerase II a negative control non immune IgG and GAPDH primers which can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol RNA polymerase II is considered to be enriched in the GAPDH gene promoter that is expected to be undergoing transcription in most growing mammalian cells and can be immunoprecipitated by RNA polymerase II antibody but not by non immune IgG In the library preparation ChlPed DNA fragments are end repaired and dA tailed end polishing simultaneously Adaptors are then ligated to both ends of the polished DNA fragments for amplification and sequencing Ligated fragments are size selected and purified using MQ binding beads which allows quick and precise size selection of DNA Size selected DNA fragments are amplified with a high fidelity PCR mix which ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimum bias Crosslink Reversal and DNA Purification n De ee o O oS Padres S Gh 43624 d NGS Illumina Fig 1 Workflow of the EpiNext ChIP Seq High Sensitivity Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368
25. ng components at 20 C immediately Enrichment Enhancer RNase A 10X End Polishing Buffer End Polishing Enzyme Mix End Polishing Enhancer 2X Ligation Buffer T4 DNA Ligase Adaptors 2X HiFi PCR Master Mix Primer U Primer I and Elution Buffer Store the following components at 4 C WB AB CB BS Protease Inhibitor Cocktail Non Immune IgG Anti RNA Polymerase II Proteinase K GAPDH Primer Forward GADPH Primer Reverse and 8 Well Assay Strips With 1 Frame and MQ Binding Beads Store all other components at room temperature All components of the kit are stable for 6 months from the date of shipment when stored properly Note Check if WB and CB contain salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Sonicator or enzymes for DNA fragmentation Vortex mixer Dounce homogenizer with small clearance pestle Variable temperature waterbath or incubator oven 0 0 0 0 O0 Thermocycler with 48 or 96 well block Page 3 Printed 2014 07 29 P 2030 Centrifuge including desktop centrifuge up to 14 000 rpm Agilent Bioanalyzer or comparable method to assess the quality of DNA l
26. nsitive DNA Library Prep Kit Illumina NGS Barcode P 1060 EpiNext NGS Barcode Index Set 12 For ChIP grade antibodies search chip grade at www epigentek com 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 20 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 07 29 Epigentek Group Inc All rights reserved Products are for research use only P 2030
27. ntol and positive control Decrease the number of PCR cycles i e 32 35 cycles to keep ampification at the exponential phase This will reduce high background in endpoint PCR and allow differences in amplification to be seen Real time PCR is another choice in such cases Little or no PCR products generated from sample wells only Poor enrichment with antibody some antibodies used in ChIP might not efficiently recognize fixed protein Increase the antibody amount and use ChIP grade antibodies validated for use in ChIP PCR primers are not optimized Confirm species specificity of primers Primers should be designed to cover a short sequence region 70 150 bp for more efficient and precise amplification of target DNA region the binding sites of the protein of interest For ChiPed DNA Library Preparation Problem Possible Cause Suggestion Low yield of library Insufficient amount of starting DNA To obtain the best results the amount of input DNA should be 100 200 ng For a library directly used for sequencing without amplification 500 ng or more is needed Insufficient purity of starting DNA Ensure that RNA is removed by RNase A treatment before starting library preparation protocol Improper reaction conditions at each reaction step Check if the reagents are properly added and incubation temperature and time are correct at each reaction step
28. only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext ChIP Seq High Sensitivity Kit and methods of use contain proprietary technologies by Epigentek 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 07 29 P 2030 A BRIEF OVERVIEW Protein DNA interaction plays a critical role for cellular functions such as signal transduction gene transcription chromosome segregation DNA replication and recombination and epigenetic silencing Identifying the genetic targets of DNA binding proteins and knowing the mechanisms of protein DNA interaction on a genome wide scale is important for understanding cellular processes Chromatin immunoprecipitation ChIP followed by next generation sequencing ChIP Seq offers an advantageous tool for studying genome wide protein DNA interactions It allows for detection that a specific protein binds to specific sequences in living cells In particular ChIP antibodies targeted against various transcriptional factors TF for genome wide transcription factor binding site analysis by Chip Seq is in high demand Such analysis requires that ChlPed DNA contain minimal background for reliably identifying true TF enriched regions Currently used ChIP Seq methods play an import
29. ort sequence region 70 150 bp for more efficient and precise amplification of the target DNA region the binding sites of the protein of interest Improper sample storage Chromatin sample should be stored at 80 C for no longer than 6 months preferably less than 3 months Avoid repeated freeze thaw cycles DNA samples should be stored at 20 C for no longer than 6 months preferably less than 3 months No difference in signal intensity between negative and positive control wells Insufficient washing Check if washing recommendations at each step is performed according to the protocol If the signal intensity in the negative control is still high washing stringency can be increased in the following ways 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 17 Printed 2014 07 29 P 2030 1 Increase wash time at each wash step after adding WB leave it in the wells for 3 4 min and then remove it 2 Add an additional one wash with WB respectively The provided volume of WB is sufficient for 4 extra washes for each sample Too many PCR cycles Plateau phase of amplification caused by excessive number of PCR cycles in endpoint PCR may mask the difference of signal intensity between negative co
30. te 3 4 pulses of 10 15 seconds each followed by 30 40 seconds rest on ice between each pulse The conditions of cross linked DNA shearing can be optimized based on cells and sonicator equipment Note When probe based sonication is carried out the shearing effect may be reduced if foam is formed in the chromatin sample solution Under this condition discontinue sonication and centrifuge the sample at 4 C at 12 000 rom for 3 min to remove the air bubbles then continue with sonication The isolated chromatin can also be sheared with various enzyme based methods Optimization of the shearing conditions for example enzyme concentration and incubation time is needed in order to use enzyme based methods c Centrifuge at 12 000 rom at 4 C for 10 min after shearing d Transfer supernatant to a new vial The chromatin solution can now be used immediately or stored at 80 C after aliquoting appropriately until further use Avoid multiple freeze thaw cycles Note The size of sonicated chromatin should be verified before starting immunoprecipitation step The length of sheared DNA should be between 100 700 bps with a peak size of about 300 bps The following steps can be carried out to isolate DNA for gel analysis of DNA fragment size 1 add 25 Ll of each chromatin sample to a 0 2 ml PCR tube followed by adding 25 ul of DRB DNA Release Buffer and 2 ul of Proteinase K 2 incubate the sample at 60 C for 30 min followed by incubating at 95
31. tforms and add the correct amount final concentration 1 5 2 uM or according to the supplier s instruction 11 Size Selection Clean up 11 1 Size Selection of Ligated DNA Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Note If the starting DNA amount is less than 50 ng the size selection is not recommended and alternatively clean up of ligated DNA can be performed prior to PCR amplification according to 11 2 protocol a Resuspend MQ Binding Beads by vortex b Add 14 ul of resuspended MQ Binding Beads to the tube of ligation reaction Mix well by pipetting up and down at least 10 times c Incubate for 5 minutes at room temperature d Put the tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully transfer the supernatant containing DNA to a new tube Caution Do not discard the supernatant Discard the beads that contain the unwanted large fragments e Add 10 ul resuspended beads to the supernatant mix well and incubate for 5 minutes at room temperature f Put the PCR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant Caution Be careful not to disturb or discard the beads that contain DNA g Keep the PCR tube in the magnetic stand and add 200 ul of freshly prepared 90 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove
32. with CB ChIP Buffer to desired concentration For histone samples containing sufficient chromatin gt 0 5 ug the Enrichment Enhancer is not required and 50 80 ul of CB ChIP Buffer can be used For low abundance targets 2 ul of Enrichment Enhancer and 88 pl of chromatin can be used without adding CB ChIP Buffer Freshly prepared chromatin can be directly used for the reaction Frozen chromatin samples should be thawed quickly at RT and then placed on ice before use Store remaining chromatin samples at 20 C or at 80 C if they will be not used within 8 hours An input DNA control is only used for estimating the enrichment efficiency of ChIP and is generally not necessary since the positive and negative control can be used for estimating the same objective more accurately If you would like to include the input DNA control the purified input DNA prepared at Step 5d Note can be used d Cap wells with strip cap and incubate at room temperature for 60 90 min on an orbital shaker 100 rpm For low abundance targets incubation time should be extended to 2 3 hours or at 4 C overnight 7 Washing of the Reaction Wells a Carefully remove the solution using a pipette and discard from each well b Wash each well with 200 ul of fresh WB each time for 4 times Allow 2 minutes on an orbital shaker 100 rpm for each wash Pipette wash buffer out from the wells c Wash each well with 200 ul of DRB one time by pipetting DRB into the well
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