EpiNext™ Post-Bisulfite DNA Library Preparation Kit (Illumina)
Contents
1. PCR tubes or plates 1 5 ml microcentrifuge tubes 100 ethanol Distilled water Oo OF OF UO O Bisulfite treated DNA sample GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiNext Post Bisulfite DNA Library Preparation Kit Illumina 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 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 Post Bisulfite DNA Library Preparation Kit Illumina is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiNext Post Bisulfite DNA Library Preparation Kit Illumina and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW2. EpiNext Post Bisulfite DNA Library Preparation Kit Illumina Base Catalog P 1055 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiNext Post Bisulfite DNA Library Preparation Kit Illumina is suitable for preparing a DNA library for various Illumina platform based bisulfite sequencing bisulfite seq assays such as whole genome bisulfite sequencing WGBS oxidative bisulfite sequencing oxBs seq reduced representative bisulfite sequencing RRBS and other bisulfite next generation sequencing applications The optimized protocol and components of the kit allow both non barcoded singleplexed and barcoded multiplexed DNA libraries to be quickly constructed using sub nanogram input concentrations of DNA since the DNA is first bisulfite converted and then used for library preparation Starting Material and Input amount Starting material is bisulfite treated DNA that is generated from various input DNA amounts ranging from 0 5 ng to 1 ug For optimal preparation the input DNA amount for bisulfite conversion should be 100 ng to 200 ng so that sufficient bisulfite treated DNA can be yielded Precautions To avoid cross contamination carefully pipette the sample or solution into the tubes vials 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 St
3. Open the cap of the PCR tube and air dry 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 2 minutes or until the solution is completely clear Transfer the clear solution to a new 0 2 ml PCR tube for the dA tailing reaction 5 DNA dA Tailing Prepare the reaction mix for dA tailing according to Table 3 Add the following reagents to a 0 2 ml PCR tube containing end repaired DNA from Step 4 Table 3 dA Tailing Reaction Component Volume End repaired DNA from Step 4 12 ul 10X dA tailing Buffer Klenow Fragment 3 5 exo Distilled Water 0 5 ul Total Volume 15 pl 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 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 Mix and incubate for 30 min at 37 C followed by 10 min at 75 C in a thermocycler 6 Adaptor Ligation Prepare a reaction mix for adaptor ligation according to Table 4 Add the following reagents to a 0 2 ml PCR tube containing end repaired dA tailing DNA from Step 5 Table 4 Adaptor Ligation Component Volume End repaired dA tailing DNA from Step 5 15 ul 2X Ligation Buffer T4 DNA Ligase
4. The prepared DNA library can be quantified using various DNA library quantification methods The prepared DNA library can be stored at 20 C until ready to use for sequencing 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 10 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 TROUBLESHOOTING Problem Possible Cause Suggestion Low yield of library Insufficient amount of bisulfite DNA Insufficient purity of bisulfite DNA Inproper reaction conditions at each reaction step Improper storage of the kit Unexpected peak size Improper ratio of MQ Binding of Agilent Beads to DNA volume during size Bioanalyzer trace selection Presence of lt 150 adaptor dimers or presence of larger fragments than expected bp Insufficient ligation Over amplification of library RELATED PRODUCTS DNA Isolation and Cleanup FitAmp General Tissue Section DNA Isolation Kit P 1003 P 1004 P 1006 P 1007 P 1009 P 1017 P 1018 FitAmp Plasma Serum DNA Isolation Kit DNA Concentrator Kit FitAmp Gel DNA Isolation Kit To obtain the best results the amount of input DNA for bisulfite treatment should be 100 200 ng Check if the sample DNA 260 280 ratio is between 1 8 1 9 and if DNA is degraded by running a gel Ensure that RNA is removed by Rnas
5. DNA methylation occurs by the covalent addition of a methyl group CH3 at the 5 carbon of the cytosine ring resulting in 5 methylcytosine 5 mC DNA methylation is essential in regulating gene expression in nearly all biological processes including development growth and differentiation Alterations in DNA methylation have been demonstrated to cause a change in gene expression For example hypermethylation leads to gene silencing or decreased gene expression while hypomethylation activates genes or increases gene expression Aberrant DNA methylation is also associated with pathogenesis of diseases such as cancer autoimmune disorders and schizophrenia Thus genome wide analysis of DNA methylation could provide valuable information for discovering epigenetic markers used for disease diagnosis and potential therapeutic targets Several methods such as whole genome bisulfite sequencing WGBS or reduced representation bisulfite sequencing RRBS are currently used for genome wide DNA methylation analysis These methods convert unmethylated cytosine to uracil while 5 mC remains unmodified by the bisulfite treatment This allows epigenetic differences to be interpreted as genetic differences which can then be detected by sequencing at single base resolution and on a genome wide scale However traditional methods to achieve this still do not have practical use because 1 such methods require large amounts of DNA gt 1 ug as input material which is dif
6. Adaptors Total Volume 34 ul Mix and incubate for 10 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 platforms and add the correct amount final concentration 1 5 2 uM or according to the supplier s instruction 7 Size Selection Clean Up 7 1 Size Selection of Ligated DNA Optional Note If the starting DNA amount is less than 200 ng size selection is not recommended and alternatively clean up of ligated DNA can be performed prior to PCR amplification according to 7 2 protocol Clean Up of Ligated DNA Resuspend MQ Binding Beads by vortex 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 Incubate for 5 minutes at room temperature 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 Add 10 ul of re suspended beads to the supernatant mix well and incubate for 5 minu
7. 56 EpiNext Bisulfite Seq High Sensitivity Kit Illumina DNA Library Preparation P 1051 EpiNext DNA Library Preparation Kit Illumina P 1053 EpiNext High Sensitivity DNA Library Preparation Kit Illumina NGS Barcode P 1060 EpiNext NGS Barcode Index Set 12 110 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 12 Printed 2013 12 31 P 1055
8. CR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant 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 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 7 2e two times for a total of three washes Open the PCR tube cap and air dry 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 2 minutes or until the solution is completely clear Transfer 11 ul of clear solution to a new 0 2 ml PCR tube for PCR amplification 8 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 Adaptor Ligated DNA 10 5 ul Total Volume 25 pl Important Note Use of Primer I included in the kit will generate a singleplexed library For multiplexed library preparation replace Primer I with on
9. No P 1026 and its Alternative Enhanced Program protocol in its user manual for DNA bisulfite treatment This kit is demonstrated to fragment DNA to about 250 bps at peak size Epigentek s Methylamp DNA modification kit Cat No P 1001 is also suitable for generating appropriate fragments that can be directly used in post bisulfite DNA library preparation 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 5 Printed 2013 12 31 P 1055 Validation of bisulfite DNA quality To ensure that the bisulfite treated DNA is suitable for direct ligation we recommend checking the bisulfite treated DNA by real time methylation specific PCR MSP For your convenience and the best results Epigentek provides the Methylamp MS qPCR Fast Kit Cat No P 1028 for real time MSP Both positive primers b actin component of kit Cat No P 1028 and negative primers GAPDH component of kit Cat No P 1029 are also separately available to verify conversion efficiency 1 dsDNA Conversion Reaction a Prepare dsDNA Conversion reaction in a 0 2 ml PCR tube according to Table 1 Table 1 dsDNA Conversion Volume Bisulfite DNA 10 ul 100 200 ng input DNA 5X Conversion Buffer 4 ul Conversion Primer Distilled Water Total Volume 19 pl Note The optimal amount of
10. are for research use only Page 4 Printed 2013 12 31 P 1055 TTTTTTTT TTT titt lcd St IIIIIIIII WIIIIIIII dsDNA Conversion m Ful g TT a dA Tailing 5 1 Ti Adaptor Ligation 5 e 7 J l Amplification Fig 2 Size distribution of library fragments as demonstrated by a post bisulfite DNA library constructed using the EpiNext Post Bisulfite DNA Library Preparation Kit from 10 ng of input DNA Fig 1 Workflow of the EpiNext Post Bisulfite DNA Library Preparation Kit Illumina ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Amount of Input DNA used for bisulfite treatment Input amount of pre bisulfite DNA can range from 500 pg to 1 ug per reaction which could yield bisulfite treated DNA ranging from at least 50 pg to 200 ng An optimal pre bisulfite DNA amount is 100 ng to 200 ng per reaction Input DNA should be of high quality and relatively free of RNA RNAse can be used to remove RNA Bisultite treated DNA Bisulfite DNA can be generated with home brew bisulfite conversion protocols or commercially available kits However most of these protocols kits cannot generate fragments of appropriate length for library preparation use The peak size of bisulfite treated DNA fragments should be 200 400 bps in length For ideal compatibility we highly recommend using Epigentek s BisulFlash DNA Modification Kit Cat
11. e 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 KIT CONTENTS Cat P 1055 12 Cat P 1055 24 Upon Receipt UserGuide E E Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped on frozen ice packs at 4 C Upon receipt store the following components at 20 C immediately 5X Conversion Buffer Conversion Enzyme Mix Conversion Primer 10X End Repair Buffer End Repair Enzyme Mix 10X dA Tailing Buffer Klenow Fragment 3 5 exo 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 MQ Binding Beads Store all other components at room temperature MATERIALS REQUIRED BUT NOT SUPPLIED Vortex mixer Agilent Bioanalyzer or comparable method to assess the quality of DNA library Thermocycler Centrifuge including desktop centrifuge up to 14 000 rpm Magnetic stand 96 well PCR plate format 0 OF OF O O0 0 Pipettes and pipette tips 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 2013 12 31 P 1055
12. e 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 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 Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 Place the reaction plate in the PCR instrument and set the PCR conditions as follows Cycle Step Temp Time Cycle 28 C 98 C 20 sec Cycling 55 C 20 sec Variable ii 20 sec Note PCR cycles may vary depending on the inout DNA amount In general use 12 PCR cycles for 200 ng 13 cycles for 100 ng 15 cycles for 50 ng 17 cycles for 10 ng and 22 cycles for 1 ng DNA input Further optimization of PCR cycle number may be required by the end user 9 Clean Up of Amplified Library DNA Resuspend MQ Binding Beads by vortex 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 Incubate for 5 minutes at room temperature to allow DNA to bind to the beads Put the PCR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant Be careful not to disturb or discard the beads that conta
13. e treatment Check if the reagents are properly added and incubation temperature and time are correct at each reaction step including Adaptor Ligation Size Selection and Amplification Ensure that the kit has not exceeded the expiration date Standard shelf life when properly stored is 6 months from date of receipt Check if the correct volume of MQ Binding Beads was added to the DNA solution accordingly Proper ratios should remove fragments of unexpected peak size Too much and too little input DNA may cause insufficient ligation which can shift the peak size of the fragment population to be shorter or larger than expected Make sure that the ligation reaction is properly processed using the proper amount of input DNA 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 FitAmp Paraffin Tissue Section DNA Isolation Kit FitAmp Urine DNA Isolation Kit FitAmp Blood and Cultured Cell DNA Extraction Kit 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 11 Printed 2013 12 31 P1055 DNA Bisulfite Conversion P 1001 Methylamp DNA Modification KIt P 1026 BisulFlash DNA Modification Kit P 10
14. ficult to prepare from limited biological samples such as tumor biopsy samples early embryos embryonic tissues and circulating DNA 2 such methods require that DNA is first sheared and then ligated to adaptors 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 followed by bisulfite conversion post ligation bisulfite conversion This procedure causes most of the DNA fragments contained in the adaptor DNA fragment constructs to be broken and thereby form mono tagged templates that will be removed during library enrichment Thus incomplete coverage and bias occur when performing whole genome bisulfite sequencing and 3 such methods are time consuming 2 days To overcome the weaknesses of these methods Epigentek offers the EpiNext Post Bisulfite DNA Library Preparation Kit Illumina The kit has the following features e Allows bisulfite converted DNA to be used directly for ligation thereby eliminating the possibility of breaking adapter ligated fragments which can often occur in currently used WGBS and RRBS methods e Fast 5 hour procedure from input starting material to library amplification e Gel free size selection purification saves time and prevents handling errors as well as loss of valuable samples e High sensiti
15. in DNA 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 Repeat Step 9e two times for total of three washes Open the PCR tube cap and air dry 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 2 minutes or until the solution is completely clear Transfer 12 ul of clear solution 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 bps at peak size without adaptors or adaptor dimers To check the size distribution dilute library 2 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 use 1X MQ Binding Beads e g dilute amplified library DNA to 20 ul with TE and then add 20 ul of MQ Binding Beads according to sub steps a through i of Step 9 Clean up of Amplified Library DNA To remove fragments above 500 bps follow sub steps a through j of Step 7 1 Size Selection of Ligated DNA
16. input DNA should be 100 ng to 200 ng and eluted volume after bisulfite treatment should be lt 20 ul b Mix and incubate for 5 min at 95 C in a thermocycler without heated lid followed by 5 min at 4 C or on ice C Add 1 ul of Conversion Enzyme Mix to the reaction tube Mix and incubate for 60 min at 37 C in a thermocycler without heated lid 2 Clean Up of Converted dsDNA a Resuspend MQ Binding Beads by vortex b Add 36 ul of resuspended beads to the PCR tube of dsDNA conversion reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times C Incubate for 10 minutes at room temperature to allow DNA to bind to the 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 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 2e one time for a total of two washes g Open the cap of the PCR tube and air dry beads for 10 minutes while the tube is on the magnetic stand h Resuspend the beads in 12 ul 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 2 minutes or until the solut
17. ion is completely clear j Transfer the clear solution to a new 0 2 ml PCR tube for the end repair reaction 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 3 DNA End Repairing Prepare end repair reaction in a 0 2 ml PCR tube according to Table 2 Table 2 End Repair Reaction Component Volume Converted dsDNA from Step 2 11 12 ul 10X End Repair Buffer End Repair Enzyme Mix Distilled Water 5 6 ul Total Volume 20 ul Mix and incubate for 30 min at 20 C in a thermal cycler 4 Clean Up of End Repaired DNA Resuspend MQ Binding Beads by vortex Add 36 ul of resuspended beads to the PCR tube of end repair reaction Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times Incubate for 10 minutes at room temperature to allow DNA to bind to the beads Put the PCR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant 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 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 4e one time for a total of two washes
18. tes at room temperature Put the PCR tube on an appropriate magnetic stand until the solution is clear about 2 minutes Carefully remove and discard the supernatant 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 80 ethanol to the tube Incubate at room temperature for 1 min and then carefully remove and discard the ethanol Repeat Step 7 1g one time for a total of two washes Open the PCR tube cap and air dry beads for 10 minutes while the tube is on the magnetic stand 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page g Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 31 Epigentek Group Inc All rights reserved Products are for research use only P 1055 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 2 minutes or until the solution is completely clear Transfer clear solution to a new 0 2 ml PCR tube for PCR amplification 7 2 Clean Up of Ligated DNA 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 P
19. vity efficiency and flexibility direct ligation of adapter to bisulfite converted DNA fragments reduces loss of fragments and selection bias which enables pre bisulfite input DNA to be as low as 1 ng The kit can be used for both non barcoded singleplexed and barcoded multiplexed DNA library preparation e Comprehensive set of components to accommodate each step of DNA library preparation ligation clean up size selection and library amplification for convenience consistency and reliability e Ultra HiFi amplification enables achievement of reproducibly high yields of DNA libraries with minimal sequence bias and low error rates PRINCIPLE amp PROCEDURE With this kit bisulfite treated DNA which is in single stranded form is converted to double stranded DNA and directly used for ligation with BisDNA specific adapters which are necessary for amplification and sequencing The fragments are then size selected and purified with MQ beads which allows for quick and precise size selections of DNA Size selected DNA fragments are then amplified with a high fidelity PCR Mix ensuring maximum yields from minimum amounts of starting material and providing a highly accurate amplification of library DNA with low error rates and minimal bias 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
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