NxSeq Long Mate Pair Library Kit Supplementary Protocol
Contents
1. Reagent Recommended Catalog Used for Blue SeaKem Vendor 2 8kb Pippin Elutrap Protocol Step 5A Step 5B SeaKem Gold Agarose Lonza 50152 No Yes Yes 50X TAE agarose gel Thermo Scientific B49 No Yes Yes running buffer Lambda DNA Hindlll NEB N3012S No Yes Yes digest Lambda DNA Mono Cut NEB N3019S No Yes Yes Mix 3 M NaOAc pH7 0 Ambion AM9740 No Yes Yes 100 Isopropanol Various Various No Yes Yes GlycoBlue Ambion AM9516 No Yes Yes 10 SDS Solution Ambion AM9822 No Yes Yes Proteinase K NEB P8107S No Yes Yes Ethidium Bromide Bio Rad 161 0433 No Yes Yes Solution at 10 mg mL 10X Loading Dye Various Various No Yes Yes 0 75 Agarose cassettes Sage Science BLF7510 No Yes No Dye Free Low Range S1 marker Sage Science i No Yes No Loading solution Sage Science No Yes No 0 1 Tween 20 Sage Science x No Yes No 3 M Sodium Acetate Life Technologies AM9740 No Yes No HpyCH4V Restriction NEB R0620S Yes Yes Yes Enzyme Rsal Restriction Enzyme R0167S Yes Yes Yes 10 U uL Alul Restriction Enzyme R0137S Yes Yes Yes 10 U uL Haelll Restriction R0108S Yes Yes Yes Enzyme 10 U L CutSmart Buffer B7204S Yes Yes Yes comes with Restriction Enzymes Accll Restriction Enzyme Takara 1002A Yes Yes Yes 10 U uL Rsal Alul Haelll Accll Life A25653 Yes Yes Yes and RE Buffer Technologies Dynabeads MyOne Life 65001 Yes Yes Yes Streptavidin C1 Technologies Agencourt AMPure XP Beckman Coulter A63881 or Yes Yes Y2. Final Recommended amount Recommended shearing conditions Desired of starting material insert size g TUBE 6200 RPM for 2 minutes each 10kb 15 Hg a ta E orientation using an Eppendorf model i 5424 Centrifuge g TUBE 5500 RPM for 2 minutes each 15kb 15 ug Sn E orientation using an Eppendorf model 5424 Centrifuge g TUBE 3200 RPM for 8 minutes each 20 kb 15 Hg es T orientation using an Eppendorf model 5424 Centrifuge 1 1 Options for shearing Covaris g TUBE Covaris Woburn MA 1 3 Size confirmation of sheared gDNA e Confirm the correct size of the sheared gDNA o Visualize on a 0 3 SeaKem Gold agarose gel in 1X TAE buffer 70 V 75 minutes Use the A Hindlll and A Monocut ladders See Figure 2 for example gel image 1 4 Quantification of Sheared gDNA e Quantify the sample from step 1 3 Purified Sheared gDNA using Qubit dsDNA HS Assay Kit with the Qubit 2 0 Fluorometer according to manufacturer s instructions Minimum amount and concentration of DNA required to proceed DNA should be in either Low TE 0 1 mM EDTA 10 mM Tris pH 8 or 10 mM Tris pH 8 5 Insert Size Minimum Amount DNA Required Minimum Concentration DNA Required 10 kb 15 kb 15 ug for g TUBE shearing 2 100 ng uL 20 kb More DNA might be needed depending on DNA shearing method O Optional Safe Stopping Point DNA can be stored at 20C Page 7 of 27 SP001 Rev A NxSeq Long M
3. 20 C for a minimum of 10 minutes up to overnight 14 16 hours e Centrifuge at 4 C for 30 minutes at 15 000 RPM e Remove supernatant being careful not to disturb the blue pellet Immediately add 600 uL of 70 Ethanol e Centrifuge at 4 C for 5 minutes at 15 000 RPM Remove supernatant with a pipette while being careful not to disturb the pellet e Air dry for 10 minutes e Carefully re suspend the pellet in 50 uL Elution Buffer EB Incubate eluted sample for 15 min at RT 5A 5 Concentration Quantify using Qubit according to manufacturer s instructions e Record the concentration in ng uL e Confirm the minimum amount and concentration of DNA required to proceed Insert Size Minimum Amount DNA Minimum Concentration DNA Required Required 10 kb 500 ng 10 0 ng uL 15 kb 750 ng 15 0 ng L 20 kb 1000 ng 20 0 ng uL SP001 Rev A Page 15 of 27 NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 5A 6 Size Confirm the correct size selection e Visualize on 0 3 SeaKem Gold agarose gel Run with Lambda DNA Hindlll digest 30 ng and Lambda DNA Mono Cut Mix 200 ng Bands at 10 15 17 and 23 kb should be visible See Figure 3 Proceed directly to step 6 Ligation of Insert to Coupler E E o a o o FO com Q at H c o v c oL v c2 Vc Zz Ona Ona Mi ZOC ZOC 242 5 bf bad 242 5 194 0 194 0 145 5 _ 2 145 5 97 0 _ 97 0 48 5 48 5 EN 2
4. NOTE Keeping the samples in the cassette for longer than 45 minutes increases the recovery of high molecular weight DNA samples from the elution modules Samples can remain in the cassette for as long as 14 16 hours overnight e Slowly extract the 40 uL size selected DNA samples from each of the two or four sample lane elution modules O Use a regular 20 200 uL tip wide bore tips will not fit in the elution module o Do not pipet the samples up and down e Combine the two or four lanes for each sample into a single 1 5 mL Lo Bind tube e _ To each of the two or four sample lane elution modules add 40 uL of 0 1 Tween 20 Page 14 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Allow the solution to sit in the elution module for 1 minute Do not pipet the sample up and down e Gently and slowly extract the 40 uL sample from each sample lane elution module and add it into the 1 5 mL Lo Bind tube for a total of 160uL for 10Kb samples and 320 uL for 15Kb and 20Kb samples 5A 4Protocol Precipitation of Captured Insert DNA In the tube with the size selected sample set up the precipitation reaction add each reagent in the following order Reagent Volume uL Volume uL 10kb insert 15 and 20kb insert Insert DNA 160 320 3 M NaOAc pH 7 0 0 1X 16 32 GlycoBlue 1 1 100 Isopropanol 1 5X 240 480 e _ Mix by inverting the tube 10 times Incubate at
5. Clean Up of Exonuclease Treated Insert Coupler In this step the Exonuclease Treated Insert Coupler from step 7 3 is cleaned 8 1 Protocol Precipitation of Exonuclease treated Insert DNA e Add the following to the sample of Exonuclease Treated Insert Coupler Reagent Volume uL per Tube Exonuclease Treated Insert Coupler 412 3 M NaOAc 0 1X 41 GlycoBlue 1 100 Isopropanol 680 e Mix by inverting the tube s 10 times Incubate at 20 C for a minimum of 10 minutes up to overnight 14 16 hours e Centrifuge at 4 C for 30 minutes at 15 000 RPM e Remove supernatant being careful not to disturb the blue pellet IMPORTANT NOTE Frequently the DNA is spread up the side of the tube and is difficult to see Always add the Elution Buffer to the side of the tube and elute the DNA by washing the side of the tube until the DNA is resuspended e Immediately add 600 uL of 70 Ethanol e Centrifuge at 4 C for 5 minutes at 15 000 RPM e Remove supernatant with a pipette while being careful not to disturb the pellet e Air dry for 10 minutes e Carefully re suspend the pellet s in a total of 35 uL Elution Buffer EB using a wide bore tip For two tubes add 17 5 uL Elution Buffer EB to each tube e Incubate eluted sample for 15 min at RT e _ For 20kb inserts combine the two tubes into a clean 1 5mL Lo bind tube for a total of 35 uL Continue with Step 11 Restriction Enzyme Digest through Step 2
6. in a cold water bath and add 15 uL of Ethidium Bromide 10 mg mL O Pour into an electrophoresis casting tray approximately 15 cm wide and 10 cm long with a comb to form 1 cm X 2 mm wells e When polymerized place agarose gel into an electrophoresis chamber containing 1X TAE buffer e Add O 10 50 uL 10X Loading Dye to the 50 uL pooled precipitated insert with ligated adaptors from step 4 4 8 ug tube O 15 and 20 kb 100 uL 10X Loading Dye to the 100 uL pooled precipitated insert with ligated adaptors from step 4 4 8 ug tube Load 50 uL sample into 2 4 internal wells 4 ug well e Load 2 0 uL each Lambda DNA Hindlll digest into two outer wells approximately 1 ug Page 17 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Run gel according to the table below Insert size Conditions 10 kb 100 Volts 60 minutes 15 kb 100 Volts 60 minutes 20 kb 70 Volts 75 minutes Note Reversing electrode polarity for 20 seconds at 1 min and 30 min into the electrophoresis can help decrease small insert contamination Excise the insert DNA band from the gel using a long wave UV hand held lamp and a single edge razor blade O Cut the agarose gel at the lower edge of the Lambda Hindlll 23 kb bands and make a second cut approximately 5 mm above the first cut O Remove the approximately 1 cm X 5 mm agarose plugs containing the insert DNA and place in a 1 5 mL tube See ex
7. 0 in the standard protocol MA160 NxSeq Long Mate Pair Library Kit User s Manual See Appendix A of this document for the expected size distribution of 20kb inserts O Optional Safe Stopping Point DNA can be stored at 20C Page 24 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Appendix A Size Distribution of 20kb Inserts Size distributions of sequenced 10 15 and 20 kb Long Mate Pair Libraries are shown below Qualified long span mate pairs were mapped against reference genomes and the pair distance distribution was plotted in CLC Genomics Workbench Paired distance distribution 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Count Figure 7 Size Distribution of 20kb Insert Size Selected with BluePippin Qualified long span mate pairs were mapped against the repeat masked reference genome GRCh38 and the pair distance distribution was plotted in CLC Genomics Workbench High Pass 20 50 kb range mode 35 kb selection 15 20 kb definition Marker S1 Paired distance distribution 45000 40000 35000 30000 25000 Count 20000 15000 10000 5000 o SS 7 Co o gt gt 2 gt a Sap o gt Soy gt O o Ro a S O S O SA A O O O O V O v O O D w Yo Do Ys Yn DM gt I gt D D SD 9 o F O Y O o 7 Q A O o Lo Lo Lo Lo Lo Lo Lo O O Lo Do Distance bp Figure 8 Size Distribu
8. 3 1 F l 23 1 9 4 A gt 9 4 6 6 6 6 4 4 4 4 E coli K12 Human Figure 3 PFGE analysis of un sheared genomic DNA Sheared DNA and Size Selection with the BluePippin for inserts 20 kb and above Page 16 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 5B Insert Size Selection using SeaKem Gold Agarose and Elutrap During this step the Precipitated Insert with Ligated Adaptor from step 4 4 is cleaned up using SeaKem Gold Agarose and Elutrap 5B 1 NxSeq Long Mate Pair Kit Box 1 Reagents Reagent Cap Identifier Elution Buffer EB 5B 2 Reagents Equipment Needed Reagent Supplied By Precipitated Insert with Ligated Adaptor From step 4 4 SeaKem Gold Agarose Lonza 50X TAE agarose gel running buffer Thermo Scientific Ethidium Bromide Solution 10 mg mL BioRad Electrophoresis supplies Various 10X Loading Dye Various Elutrap Whatman 3 M NaOAc pH7 0 Ambion GlycoBlue Ambion 100 Isopropanol User 1 5 mL LoBind Microcentrifuge tubes Eppendorf 5B 3 Protocol Agarose Gel Size Selection Prepare a SeaKem Gold agarose gel O Add SeaKem Gold Agarose to 1X TAE according to the table below Insert size SeaKem Gold 1X TAE mL Agarose g 10 kb 1 0 100 15 kb 0 5 100 20 kb 0 3 100 O Heat the TAE and agarose mixture to boiling to dissolve agarose O Cool the solution for 10 sec
9. Centrifuge at Room Temperature for 5 minutes at 15000 RPM e Remove supernatant with a pipette while being careful not to disturb the pellet e Air dry for 10minutes e For each tube resuspend the pellet in O 60 uL Elution Buffer EB with a wide bore tip for the BluePippin protocol in Section 5A O 50 uL Elution Buffer EB with a wide bore tip for the SeaKem Elutrap protocol in Section 5B Incubate the eluted sample for 15 min at Room Temperature e Centrifuge at high speed 12 000 15 000 rpm for 5 minutes to remove insoluble material e Transfer and pool the supernatants into a single clean 1 5 mL LoBind tube Page 12 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 5A Size select the Adaptor Ligated DNA with BluePippin During this step the Precipitated Insert with Ligated Adaptor from step 4 4 is cleaned up using the BluePippin 5A 1 NxSeq Long Mate Pair Kit Box 1 Reagents Reagent Cap Identifier Elution Buffer EB 5A 2 Reagents Equipment Needed Reagent Supplied By Precipitated Insert with Ligated Adaptor From step 4 4 BluePippin Sage Science 0 75 Agarose cassettes Dye Free Low Range Sage Science S1 marker Sage Science Loading solution Sage Science 0 1 Tween 20 Sage Science 3 M Sodium Acetate Ambion 100 Isopropanol User 70 Ethanol Prepare fresh daily User 1 5 mL LoBind Microcentrifuge tubes Eppendorf Ethidium Bromide S
10. ME Lucigen Simplifying Genomics NxSeq Long Mate Pair Library Kit Supplementary Protocol Generating Libraries using 10 20kb inserts FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE Lucigen Corporation 2905 Parmenter St Middleton WI 53562 USA Toll Free 888 575 9695 608 831 9011 FAX 608 831 9012 lucigen lucigen com www lucigen com SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Product Description The NxSeq Long Mate Pair Library Kit is designed to generate mate pair libraries for sequencing on Illumina platforms This supplementary protocol describes the use of the NxSeq Long Mate Pair Library Kit reagents to generate mate pair libraries using 10 20kb insert sizes When combined with fragment library sequencing data mate pair library sequences enable superior genome assembly closure and finishing Applications include de novo genome assembly chromosomal rearrangement detection haplotyping and BAC sequencing In order to generate 10 15 and 20kb libraries the following reagents are required Catalog Description Kit Size 13000 1 NxSeq Long Mate Pair Library Kit 5 libraries gt 13100 1 NxSeq Long Mate Pair Library and 5 libraries Index Kit 12 indices 5 libraries each 13200 1 NxSeq Long Mate Pair Library Index 12 indices 5 libraries each Kit 13300 1 NxSeq Long Mate Pair Library Kit 5 libraries Box 1 13400 1 NxSeq Lo
11. T NOTE For 10 and 15kb insert ligations the insert will be ligated in a single tube For 20kb insert ligations the insert will be split into two tubes with 500ng in each tube e Inasingle fresh 1 5 mL LoBind tube 10 and 15 kb or two fresh 1 5 mL LoBind tubes 20kb set up the following ligation reactions to generate the Ligated Insert Coupler add each reagent in the following order Reagents for 10 and 15 kb inserts Tube 1 Volume uL Size selected Insert with adaptor x calculated in step 6 3 2 Coupler mix CM 3 0 Nuclease free water Up to 356 5 10X Ligase Buffer 10X 40 Ligase LIG 0 5 Total 400 Reagents for 20 kb inserts Tube 1 Volume uL Tube 2 Volume uL Size selected Insert X 2 X 2 with adaptor 500 ng calculated in step 6 3 2 calculated in step 6 3 2 each reaction Coupler mix CM 1 5 1 5 Nuclease free water Up to 356 5 Up to 356 5 10X Ligase Buffer 10X 40 40 Ligase LIG 0 5 0 5 Total 400 400 NOTE Use a pipet designed for volumes under 2 uL to pipet the Ligase e _ Mix gently by inverting tube s 10 times e Incubate and heat kill the Ligated Insert Coupler from step 6 4 according to the table below Step Temperature Time 1 4 5 C Overnight 14 16 hours 2 70 C 15 minutes Place the tube s on ice for 2 minutes Spin the tube s briefly to collect materials at the bottom of the tube s Proceed directl
12. ach of the following lanes O For 10kb insert Two of the four remaining lanes O For 15 and 20kb inserts Four remaining lanes NOTE This protocol is optimized for 3 4 ug of DNA loaded per lane If your amount of input DNA is outside of this range additional optimization may be required e _ Under the Protocol Editor tab program the BluePippin run with the following parameters O Range Mode Settings see table below O BluePippin Cassette Definition see table below O Indicate the reference lane loaded with the S1 marker by choosing the appropriate flag and select apply reference to all lanes O For the four sample lanes select the range box O For the four sample lanes enter the desired start value and a desired end value from the table below Minimum Range Mode Settings BluePippin Cassette Definition Insert Size 10kb Bpstart 10000 Bpend 50000 9 75 DF Marker S1 high pass 6 10kb vs3 15 kb Bpstart 15000 Bpend 50000 9 75 DF Marker S1 high pass15 20 kb 20 kb Bpstart 20000 Bpend 50000 9 759 DF Marker S1 high pass15 20 kb e Save the parameters as a named pprot file e Under the Main tab click on start The required run time is dependent on the minimum insert size For example 10kb insert will take approximately 2 hours whereas a 20kb insert will take up to 4 5 e After the run ends allow the size selected samples to sit in the cassette for a minimum of 45 minutes
13. ample in Figures 4 and 5 20 kb 15 kb 10 kb S e Se g e E E E 3 e e x sx ut 2 M 0 3 0 5 1 0 Figure 4 Zone of Compression ZOC Inserts located in the Zones of Compression are excised for elution using the Elutrap dd d dl led aa gt 23kb taa Figure 5 Gel Isolated Insert Sheared adapted insert was gel isolated on a 0 3 SeaKem Gold agarose gel The marker is a Lambda Hindlll digest The lower bands are un ligated adaptor Page 18 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 5B 4 Protocol Elutrap Capture of Insert DNA e Setup the Elutrap according to manufacturer s instructions Place two agarose plugs in the Elutrap next to the white BT2 filter stacked horizontally or vertically side by side 15 and 20 kb libraries require 2 Elutraps each e Apply 150 Volts for 5 hours e After electrophoresis reverse the polarity of the electrodes and apply 200 Volts for 20 seconds to remove DNA from the BT1 membrane e Remove the buffer containing the insert from the sample chamber with a 1000 mL pipette tip and place into a fresh 1 5 mL LoBind tube approximately 400 to 500 mL Take care not to puncture the fragile white BT2 membrane Note The amount of sample removed will be variable 5B 5 Protocol Precipitation of Captured Insert DNA e Measure the volume of the sample removed from the sample chamber X in table below Split into two tubes
14. ate Pair Library Kit 10 20kb Insert Libraries 10000 6000 4000 3000 2000 1000 High mass AHindill Lambd 28 kb G Tube Shear La A Mono Shear A Shear B a pp RE 10 3 AHindi l High mass Kb 23 1 44 6 6 4 4 Figure 2 Sheared genomic DNA Genomic DNA was sheared to approximately 28 kb with a g TUBE and visualized on a 0 3 SeaKem Gold agarose gel Markers include High Mass Ladder Lambda Hindlll Lambda genomic DNA and Lambda Mono Cut Mix SP001 Rev A Page 8 of 27 NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 2 End Repair During this step the sheared gDNA from step 1 is end repaired Each end repair reaction is limited by the number of DNA molecules Therefore the number of reactions performed at this step is determined by the insert size Insert Size Recommended of reactions 10 kb 8 938 ng each reaction 15 kb 16 938 ng each reaction 20 kb 16 938 ng each reaction 2 1 NxSeq Long Mate Pair Kit Box 1 Reagents Reagent Cap Identifier End Repair Tailing Buffer ERB End Repair Enzyme Mix ERE 2 2 User Supplied Reagents Equipment Reagent Supplied By Purified Sheared gDNA From step 1 4 Nuclease Free Water Ambion 0 2 mL thin wall PCR tubes Eppendorf Thermocycler User 2 3 Protocol e Add the following reagents to 0 2 mL thin wall PCR tubes number of reactions determined in
15. below Insert Size Minimum Amount DNA Minimum Concentration DNA Required Required 10kb 500 ng 10 0 ng uL 15 kb 750 ng 15 0 ng uL 20 kb 1000 ng 20 0 ng uL 5B 6 Size Confirm the correct size selection e Visualize on 0 3 SeaKem Gold agarose gel Run with Lambda DNA Hindlll digest 30 ng and Lambda DNA Mono Cut Mix 200 ng The 10 15 17 and 23 kb bands should be visible See Figures 6 and 7 Proceed directly to step 6 Ligation of Insert to Coupler High 5 A Hind3 A DNA Mono Genomic A Hind3 High 5 Insert _ npe f n i 10 1 10000 ltt Figure 6 Gel Isolated Insert DNA 20 kb insert DNA was gel isolated on a 0 3 SeaKem Gold agarose gel and eluted with an Elutrap electro eluter Purified insert DNA was visualized on a 0 3 SeaKem Gold agarose gel Markers include High Mass Ladder Lambda Hindlll Lambda genomic DNA and Lambda Mono Cut Mix ass 97 32 63 5 mw 48 5 33 5 i 23 1 15 9 42 Page 20 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Figure 7 Optional Method for Visualization FIGE analysis of purified insert DNA Insert DNA arrow was separated on a 0 6 agarose gel by Field Inversion Gel Electrophoresis using a BioRad CHEF DR Ill System 6 Ligation of Insert to Coupler During this step the size selected DNA with adaptor from step 5A 6 or 5B 6 is ligated to the coupler 6 1 NxSeq Long Mate Pair Kit Box 2 Rea
16. een optimized for the best performance o The materials provided in the kit are quantified using Qubit 2 0 Fluorometer Life Technologies o The use of other quantification methods e g gel image A260 A280 may lower the efficiency of the kit and result in insufficient material to sequence Detailed Protocol 1 Shear DNA to Appropriate Size During this step the genomic DNA gDNA is sheared to an average size range that is larger than the desired insert size See Appendix B The effect of the size range of sheared DNA on insert size for additional information on shearing and size selection Notes e DNA used must be free of contaminating RNA e gDNA used must be of a high molecular weight gt 20 kb e gDNA must be resuspended in Low TE 0 1 mM EDTA 10 mM Tris pH 8 or in 10 mM Tris pH 8 5 e Tagmentation from Illumina should not be used for shearing Tagmentation will add additional nucleotides and the use of Tagmentation has not been tested with the mate pair kit Page 6 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries A sample loss of 20 60 is expected during shearing and clean up The percentage of sample loss will vary depending on the shearing method used This expected loss should be taken into account when determining the amount of gDNA to shear Use the table below to determine the recommended amount of starting gDNA and shearing method for your final desired insert size
17. es Magnetic Beads A63882 Page 4 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Reagent Recommended Catalog Used for Blue SeaKem Vendor 2 8kb Pippin Elutrap Protocol Step 5A Step 5B 100 Ethanol Various Various Yes Yes Yes Nuclease Free Water Ambion AM993 Yes Yes Yes not DEPC treated 1 5 mL Eppendorf DNA Eppendorf 22431021 Yes Yes Yes LoBind Microcentrifuge tubes 0 2 mL thin wall PCR Various Various Yes Yes Yes tubes Qubit dsDNA HS Assay Invitrogen Q32854 Yes Yes Yes Kit Bioanalyzer DNA Kits Agilent 5067 4626 Yes Yes Yes Options include Technologies 5067 1508 e Agilent High Sensitivity DNA Kit e Agilent DNA 12000 Kit optional Included with 0 75 Agarose cassettes Dye Free Low Range Equipment Recommended Catalog Used for Blue SeaKem Vendor 2 8kb Pippin Elutrap Protocol 5A 5B Wide Bore Pipet Tips Axygen TF 205 WB L No Yes Yes 200 uL R S Wide Bore Pipet Tips Axygen TF 1005 WB No Yes Yes 1000 uL R S Eppendorf Centrifuge Eppendorf 5424 No Yes Yes Electrophoresis supplies Various Various No Yes Yes e SeaKem Gold Lonza 50152 No Yes Yes Agarose Lucigen 50020 1 or e Markers 1K plus and NEB 50010 1 100 bp NEB N3012S e Marker Lambda DNA N3019S Hindlll digest e Marker Lambda DNA Mono Cut Mix Covaris g TUBE Covaris 520079 or No Yes Yes 520104 Refri
18. g parameters Step Temperature Time 1 37 C 20 minutes 2 70 C 15 minutes 3 4 C Hold Proceed directly to step 4 Ligation of Adaptor SP001 Rev A Page 10 of 27 NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 4 Ligation of Adaptor During this step the A tailed gDNA from step 3 3 is ligated to the adaptor 4 1 NxSeq Long Mate Pair Kit Box 1 Reagents NOTE Do not vortex the adaptor Mix by pipetting up and down and spin down briefly prior to use Reagent Cap Identifier Adaptor ADT Ligase LIG 4 2 User Supplied Reagents Equipment Reagent Supplied By A tailed gDNA From step 3 3 10 SDS Solution Ambion Proteinase K NEB 3 M NaOAc pH7 Ambion 100 Isopropanol Various GlycoBlue Ambion Thermocycler User 4 3 Ligation e Inthe tube with the A tailed gDNA set up the ligation reactions add each reagent in the following order Reagent Volume HL for each reaction A tailed gDNA 52 Adaptor ADT 6 Ligase LIG 4 Total 62 e Mix by pipetting up and down 10 times with a wide bore tip Place tube in the a thermocycler and incubate according to the following parameters Step Temperature Time 1 25 C 30 minutes 2 4 C Hold Pool ligation reactions o For 10kb libraries pool reactions 1 8 into clean 1 5 mL LoBind tubes Spin the tubes briefly to collect materials at t
19. gents Reagent Cap Identifier Coupler Mix CM 10X Ligase Buffer 10X Ligase LIG 6 2 User Supplied Reagents Equipment Reagent Supplied By Size selected DNA with Adaptor From step 5A 6 or 5B 6 Nuclease Free Water Ambion Refrigerator set at 4 5 C Various Thermomixer or heat block set at 70 C Eppendorf Pipet designed for volumes under 2uL Various 6 3 Determine Amount of Insert Required Use the following equation to determine the amount of size selected DNA with adaptor material is required for step 6 4 Protocol NOTE The optimal condition for this step is to use equal amounts of insert and coupler in the ligation reaction The size of the coupler included in the kit is 2000 bp and the amount of coupler specified for each reaction is 100 ng insert size bp 2000 bp x100 ng ng insert Y 5000 bp 2000 bp Example x100ng 250ng 6 3 1 Calculate and record the amount of insert required e Use the following equation to determine the required volume of size selected DNA with adaptor required X based on the concentration determined in step 5A 5 or 5B 5 Z and the amount of insert Y in ng as calculated above Y Amount in ng ng X volume in ul Z Concentration in IL 6 3 2 Calculate and record the volume of insert required X volume in uL Page 21 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 6 4 Protocol amp IMPORTAN
20. gerator 4C Various Various No Yes Yes calibrated to 4 5 C BluePippin Sage Science n a No Yes No Elutrap Whatman 10 447 700 No No Yes or Long wave UV 365 nM UVP Inc Model UVL No No Yes Blak Ray Lamp 56 Page 5 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Equipment Recommended Catalog Used for Blue SeaKem Vendor 2 8kb Pippin Elutrap Protocol 5A 5B 2100 Bioanalyzer Agilent Various Yes Yes Yes Electrophoresis supplies Various Various Yes Yes Yes ici Lucigen 50020 1 or Yes Yes Yes e Markers 1K plus and 50010 1 100 bp Prior to starting Restriction Enzyme Selection Before proceeding with library construction you must identify the restriction enzyme s needed to digest the gDNA to 400 900 bp desired final library after PCR amplification This step is critical to ensure the kit performs as designed and the sequencing coverage is uniform Refer to this section in the standard protocol MA160 NxSeq Long Mate Pair Library Kit User s Manual for detailed instructions General Recommendations Use Eppendorf Lo Bind 1 5 mL tubes throughout the protocol Thaw all kit reagents on ice prior to use Use wide bore tips to handle High Molecular Weight DNA Use a Qubit Fluorometer or equivalent to perform all sample quantification throughout the protocol o The ratios of material used in each ligation step throughout the protocol have b
21. he bottom of the tubes o For 15 and 20kb libraries pool reaction 1 8 and 9 16 into two clean 1 5 mL LoBind tubes SP001 Rev A Page 11 of 27 NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries e Calculate the total volume of pooled ligation reactions and record the value Reagent Volume HL Volume HL Volume HL for each reaction reactions 1 8 reactions 9 16 A tailed gDNA 62 496 496 10 SDS Solution 4 32 32 Proteinase K 4 32 32 Total 70 560 560 e Mix by inverting tube 10 times Spin briefly to collect material in the bottom of the tube Place tube in the thermomixer or heat block and incubate according to the following parameters Step Temperature Time 1 37 C 30 minutes 4 4 Precipitate Ligated Material e Inthe tube containing the material treated with Proteinase K set up the precipitation reactions add each reagent in the following order Reagent Volume uL Volume uL reactions 1 8 reactions 9 16 Proteinase K treated DNA 560 560 3 M NaOAc 56 56 GlycoBlue 1 1 100 Isopropanol 900 900 e _ Mix by inverting tube 10 times Incubate at Room Temperature for 10 minutes e Centrifuge at Room Temperature 25 C for 30 minutes at 15000 RPM Note SDS will precipitate at lower temperatures e Remove supernatant being careful not to disturb the blue pellet Immediately add 600 uL of 70 Ethanol e
22. if the volume of the mixture exceeds 1 5 mL e Add the following to the tube s containing the insert DNA Reagent Volume Example 1 Example 2 Volume HL Volume HL Gel Excised Insert DNA step 5B 3 X 400 500 3 M NaOAc pH 7 0 0 1X 0 1 X 40 50 GlycoBlue 1 uL 1 1 100 Isopropanol 1 5X 1 5X 660 825 Mix by inverting the tube 10 times Incubate at 20 C for a minimum of 10 minutes up to overnight 14 16 hours Centrifuge at 4 C for 30 minutes at 15 000 RPM Remove supernatant being careful not to disturb the blue pellet Immediately add 600 uL of 70 Ethanol Centrifuge at 4 C for 5 minutes at 15 000 RPM Remove supernatant with a pipette while being careful not to disturb the pellet Air dry for 10 minutes Carefully re suspend the pellet s using the following amounts of Elution Buffer EB O Single Tube 50 uL O Two Tubes 25 uL in each tube Incubate eluted sample for 15 min at RT If sample is split into two tubes and resuspended in 25 uL of Elution Buffer EB pool tubes into clean 1 5mL tube Page 19 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries e Combine 15 and 20 kb insert Elutrap samples 2 Elutraps each 5B 5 Concentration Quantify using Qubit according to manufacturer s instructions e Record the concentration in ng L e Confirm the minimum amount and concentration of DNA required to proceed using the table
23. ng Mate Pair Library Kit 10 15kb 10 libraries Box 2 20kb 6 libraries 1 The reagents in box 1 are sufficient for 5 libraries using 10 20kb inserts 2 The reagents in box 2 are sufficient for 10 libraries using 10 15kb inserts 3 The reagents in box 2 are sufficient for 6 libraries using 20kb inserts Supplementary Protocol Workflow This supplementary protocol replaces steps 1 10 in the User s Manual MA160 After the completion of the protocol outlined in this document users will return to the protocol in the User s Manual MA160 The restriction enzyme testing described in the User s Manual is required prior to starting the 10 20kb protocol outlined in this document This supplementary protocol describes the use of two methods for step 5 Size Selection these methods include BluePippin Isolation and Gel Isolation using SeaKem Gold Agarose gels and Elutraps Step 1 Shearing through step 4 Ligation of Adaptor and Step 6 Ligation of Insert to Coupler through Step 8 Clean up are the same regardless of the size selection method Refer to this section in the standard protocol MA160 NxSeq Long Mate Pair Library Kit User s Manual for additional details on the overall mate pair library workflow Page 2 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Components and Storage Store all kits and components at 20 C 5 C max 25 C min 1 NxSeq Long Mate Pai
24. olution 10 mg mL BioRad 10X Loading Dye Various 5A 3 BluePippin Size Selection e Prepare the 0 75 agarose dye free cassette according to the following instructions O Gently tap any bubbles out from behind the elution modules O Remove electrophoresis buffer from all five elution modules and replace it with fresh electrophoresis buffer O Seal the elution modules shut with the provided tape O Ensure that the buffer level is sufficient in all chambers O Fill each sample well completely with electrophoresis buffer and then remove 40 uL from each well O Calibrate the instrument and test the cassette Do not use any lanes that fail e Prepare the insert for loading onto the BluePippin O Add 10 kb 20 uL of BluePippin loading solution to the tube containing 60 uL of resuspended insert DNA Do not vortex to mix 15 and 20 kb 40 uL of BluePippin loading solution to the tube containing 120 uL of resuspended insert DNA Do not vortex to mix O Mix by pipetting up and down about 10 times with a wide bore pipet tip until the sample is equilibrated with loading solution O Spin the tube briefly to collect materials at the bottom of the tubes e Load the cassette with 40 uL of S1 marker in one lane designated as the reference lane Page 13 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Using a wide bore tip load 40 uL of the resuspended insert DNA plus loading solution into e
25. r Kit Box 1 Reagent Name tubes in kit Cap Map Identifier A943016 Identifier Elution Buffer 1 EB EB Elution Buffer End Repair Tailing Buffer 2 ERB ERB E R Buffer End Repair Enzyme Mix 1 ERE ERE E R Enzyme Klenow Fragment 1 KF KF Klenow Adaptor 1 ADT ADT Adaptor Ligase 1 LIG Lig Ligase Reagents only in Box 1 NxSeq Long Mate Pair Kit Box 2 Reagent Name tubes in kit Cap Map Identifier A943018 Identifier Elution Buffer EB 5 EB EB Elution Buffer Klenow Fragment 1 KF KF Klenow Ligase 1 LIG Lig Ligase Coupler Mix 1 CM CM Coupler 10X Ligase Buffer 1 10X 10X Ligase Buffer Nuclease 1 1 N1 N1 Nuclease 1 Nuclease 2 1 N2 N2 Nuclease 2 Biotin Wash Buffer 4 BWB BWB Biotin Wash Biotin Capture Buffer 1 BCB BCB Biotin Buffer Biotin Capture Reagent 1 BCR BCR Biotin Reagent Tailing Buffer 1 TB TB Tailing Buffer Junction Code Reagent 1 JC JC Junction Code T4 Polynucleotide Kinase 1 PNK PNK Accura HotStart 2X Master Mix 1 AMM AMM Accura 2X MM Primer Mix Index 12 1 12 12 Index 12 Page 3 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Customer Supplied Reagents and Equipment Note The customer supplied reagents and equipment vary depending on the size selection method
26. table in step 2 End Repair Reagent Amount for each reaction Purified sheared gDNA 938 ng Nuclease Free Water Up to 23 uL End Repair Tailing Buffer ERB 25 uL End Repair Enzyme Mix ERE 2 uL Total 50 uL Mix by pipetting up and down 10 times using a wide bore tip Place tube s in a thermocycler and incubate according to the following parameters Step Temperature Time 1 25 C 20 minutes 2 72 C 25 minutes 3 4 C Hold Proceed directly to step 3 A Tailing Page 9 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 3 A Tailing During this step the End Repaired gDNA from step 2 3 is A tailed The number of reactions performed during this step is the same as the number of reactions performed in step 2 End Repair 3 1 NxSeq Long Mate Pair Kit Box 1 Reagents Reagent Cap Identifier Klenow Fragment KF 3 2 User Supplied Reagents Equipment Reagent Supplied By End repaired gDNA From step 2 3 Thermocycler User 3 3 Protocol Using the tubes containing the End repaired sheared DNA set up the A tailing reaction add each reagent in the following order Reagent Volume HL for each reaction End repaired gDNA 50 Klenow Fragment KF 2 Total 52 Mix by pipetting up and down 10 times with a wide bore tip Place the tube in a thermocycler and incubate according to the followin
27. tion of 20kb Insert Size Selected with Elutrap Sequencing data was mapped against the reference E coli strain K12 and plotted as Paired Distance Distribution Page 25 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit Paired distance distribution 10 20kb Insert Libraries 90000 80000 70000 60000 3 50000 40000 30000 20000 10000 0 Oo tb vs katana 0 DO G A S GQ S KA N LT O E V V O D 9 I A Y di Y 2 2 2 2 O Qa Y e Y o Y ZH Yen O o Go A Oo Y YY e Y Y Distance bp Figure 9 15 kb Mate Pair E coli Elutrap Paired distance distribution 90000 80000 70000 60000 50000 23 o Y 40000 30000 20000 10000 0 Oo os So 25 Yo Ya Ys Y gt gt 2 gt gt 7 o Q w Y GQ Y 0 SS a S g ae pen O 5 q O O O 9 27 amp e g e gt e Y o 9 Ba o S gt OQ DS gt gt Y Y 9 Z 9 2 9 Z 9 Y Y Y DY Y Y Y as Distance bp Figure 10 10 kb Mate Pair E coli Elutrap Page 26 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries Paired distance distribution 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 Count Distance bp Figure 11 20 kb Mate Pair Thermus aquaticus Elutrap Page 27 of 27 SP001 Rev A
28. y to Step 7 Exonuclease Treatment Page 22 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 7 Exonuclease Treatment During this step the Heat Killed Ligated Insert Coupler from step 6 4 is treated to remove any linear DNA 7 1 NxSeq Long Mate Pair Kit Box 2 Reagents Reagent Cap Identifier Nuclease 1 N1 Nuclease 2 N2 7 2 Reagents Equipment Needed Reagent Supplied By Heat Killed Ligated Insert Coupler From Step 6 4 Thermomixer or heat block set at 37 C Eppendorf Thermomixer or heat block set at 80 C Eppendorf 7 3 Protocol e Inthe tube s with the Heat Killed Ligated Insert Coupler set up the exonuclease treatment add each reagent in the following order Reagent Volume uL per Tube Heat Killed Ligated Insert Coupler 400 Nuclease 1 N1 7 Nuclease 2 N2 5 Total 412 e Mix gently by pipetting up and down 10 times with a wide bore tip e Place tube s in a thermomixer or heat block and incubate according to the following parameters Step Temperature Time 1 37 C 30 minutes 2 80 C 30 minutes e Place the tube s on ice for 2 minutes e Spin the tube s briefly to collect materials at the bottom of the tube s e Proceed directly to Step 8 Clean up of Exonuclease Treated Insert Coupler Page 23 of 27 SP001 Rev A NxSeq Long Mate Pair Library Kit 10 20kb Insert Libraries 8
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