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TruSeq Nano DNALibrary Prep for NeoPrep Reference GuideFor Research Use Only. Not for use in diagnostic procedures.Revision HistoryIntroductionAdditional ResourcesDNA Input RecommendationsPipette and Tip RequirementsTips and TechniquesLibrary Prep WorkflowSelect Samples and IndexesFragment DNAPrepare Samples for LoadingSet Up Run and Load Library CardUnload Libraries[Optional] Validate Libraries Manually[Optional] Normalize Libraries ManuallyPool LibrariesSupporting InformationTechnical AssistanceILLUMINA PROPRIETARYCatalog # NP-101-9001DOCMaterial # 20000943Document # 15049722 v01October 20153456781113141618242628293037
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for thecontractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. Thisdocument and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed,or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any licenseunder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in orderto ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully readand understood prior to using such product(s).FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREINMAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, ANDDAMAGE TO OTHER PROPERTY.ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE). 2015 Illumina, Inc. All rights reserved.Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect,MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA, TruGenome,TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and thestreaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All othernames, logos, and other trademarks are the property of their respective owners.AMPure , Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.
DocumentMaterial # 20000943Document #15049722 v01DateOctober2015Description of ChangeChanged loading order to load samples firstChanged loading workflow on reagent plate guide and library cardguideAdded 3 minute wait to drain oil from vial into library cardRenamed button Home at the conclusion of unloading librariesConsumables table: Removed 20 µl and 200 µl pipettes. They are specified in Pipettesand Tips. Removed 1000 µl pipettes as they are standard lab itemsMoved Acronyms to end of Supporting InformationPart # 15049722 Rev. CJune2015Added required pipettes to: Pipette Tip Requirements Consumables and EquipmentAdded step to vortex DMB in Prepare Samples for LoadingRemoved part numbers and package layouts from Kit ContentsUpdated reagent names in Reagent Plate ContentsPart # 15049722 Rev. BApril2015Added required pipette tip and calibration information to: New Pipette Tip Requirements Consumables and Equipment Tips and Techniques Load the Library CardChanged BaseSpace resource reference to helpcenterPart # 15049722 Rev. AMarch2015Initial release.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide3Revision HistoryRevision History
IntroductionThis protocol explains how to prepare up to 16 indexed paired-end libraries of genomicDNA (gDNA) using the reagents provided in the Illumina TruSeq Nano DNA LibraryPrep Kit for NeoPrep . The purpose of the protocol is to add adapter sequences onto theends of DNA fragments. The result is indexed paired-end libraries for single read orpaired-end sequencing. The libraries are ready for subsequent cluster generation andsequencing.The protocol offers:} Streamlined workflow} All reagents required for library prep, quantification, and normalization are included} 30 minutes hands-on time} Optimized shearing for whole-genome resequencing with 350 bp and 550 bp insertsize workflows} A disposable library card allowing for simultaneous preparation of up to 16 DNAsamples} Use of 16 default index adapters, plus 8 alternate index adapters, allowing up to24-plex pooling with additional NeoPrep runs} Universal adapters for preparation of single read, paired-end, and indexed libraries4Material # 20000943Document # 15049722 v01
Additional ResourcesAdditional ResourcesThe following documentation is available for download from the Illumina website.ResourceDescriptionTruSeq Nano DNA Library Prepfor NeoPrep Protocol Guide(document # 15059579)Provides only protocol instructions.The protocol guide is intended for experienced users.TruSeq Nano DNA Library Prepfor NeoPrep Checklist (document #15068125)Provides a checklist of the protocol steps.The checklist is intended for experienced users.NeoPrep Library Prep SystemGuide (document # 15049720)Provides an overview of instrument components andsoftware, instructions for performing library prep runs, andprocedures for proper instrument maintenance andtroubleshooting.Illumina Experiment ManagerGuide (document # 15031335)and IEM NeoPrep QuickReference Card (document #15061111)Provide information about creating and editing appropriatesample sheets for Illumina sequencing systems and analysissoftware and record parameters for your sample plate.BaseSpace help(help.basespace.illumina.com)Provides information about the BaseSpace sequencing dataanalysis tool that also enables you to organize samples,libraries, pools, and sequencing runs in a singleenvironment.TruSeq Library Prep PoolingGuide (document # 15042173)Provides TruSeq pooling guidelines for preparing librariesfor Illumina sequencing systems that require balanced indexcombinations. Review this guide before beginning librarypreparation.Visit the TruSeq Nano DNA Library Prep Kit for NeoPrep and NeoPrep System supportpages on the Illumina website for requirements and compatibility, additionaldocumentation, software downloads, online training, frequently asked questions, andbest practices.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide5
DNA Input RecommendationsFor best results, follow the input recommendations. Quantify the input gDNA and assessthe gDNA quality before beginning library preparation.} For a 350 bp insert size, use 25 ng input gDNA. Do not use more than 50 ng gDNA.} For a 550 bp insert size, use 75 ng input gDNA. Do not use more than 150 ng gDNA.} Input amounts lower than those specified results in low yield and increasedduplicates.Quantify Input DNAUse the following recommendations to quantify input DNA:} Successful library preparation depends on accurate quantification of input DNA. Toverify results, use multiple methods.} Use fluorometric-based methods for quantification, such as Qubit or PicoGreen.} DNA quantification methods that rely on intercalating fluorescent dyes measure onlydouble-stranded DNA and are less subject to the presence of excess nucleic acids.} Do not use spectrophotometric-based methods, such as NanoDrop, which measurethe presence of nucleotides and can result in an inaccurate measurement of gDNA.} Quantification methods depend on accurate pipetting methods. Do not use pipettesat the extremes of volume specifications. Make sure that pipettes are calibrated.Assess DNA QualityAbsorbance measurements at 260 nm are commonly used to assess DNA quality:} The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indicationof sample purity. Values of 1.8–2.0 indicate relatively pure DNA.} The presence of RNA or small nucleic acid fragments, such as nucleotides, cancompromise both absorbance measurements.} Make sure that samples are free of contaminants.Positive ControlIllumina recommends using Coriell Institute gDNA (NA 12878) as a positive controlsample for this protocol.6Material # 20000943Document # 15049722 v01
Use the following required pipettes and tips. Other pipettes and tips are not supportedand can result in reagents not dispensing properly and run failure.Table 1 Required User-Supplied Pipettes and TipsVolume20 µl200 µlUse 20 µlProduct NameSupplierPipet-Lite XLS 8-channel LTS, 2 µl to 20 µl Rainin,catalog # L8-20XLS One of the following: LTS tips 20 µl. Presterilized. Filter Rainin,catalog # RT-L10F Fisher Scientific, ART Barrier Pipette Tips 20 µl; 20 µlcatalog # 2749RISoftFit-L21–200 µl Pipet-Lite XLS 8-channel LTS,Rainin,20 µl to 200 µlcatalog # L8-200XLS One of the following: LTS tips 200 µl. Presterilized. Filter Rainin,catalog # RT-L200F Fisher Scientific, ART Barrier Pipette Tips 200 µl; 200 µlcatalog # 2769RISoftFit-LTruSeq Nano DNA Library Prep for NeoPrep Reference Guide7Pipette and Tip RequirementsPipette and Tip Requirements
Tips and TechniquesSealing a Plate}}}Always seal the 96-well plate before the following steps in the protocol:} Shaking} Vortexing} CentrifugeApply the adhesive seal to cover the plate and seal with a rubber roller.Microseal 'B' adhesive seals are effective at -40 C to 110 C, and suitable for skirted orsemiskirted PCR plates.Covaris Tubes and Instruments}}}}}}Make sure that pipettes are calibrated before beginning. Uncalibrated pipettes canlead to variations in insert size, and reagents not dispensing properly, resulting inrun failure.To ensure reproducible DNA shearing, centrifuge samples before fragmenting.Load DNA samples into the Covaris tube slowly to avoid creating air bubbles.However, air bubbles might not be preventable.Use the wells of a plate or another device to hold the Covaris tubes upright.Remove the Intensifier from the E220 instrument before fragmenting with amicroTUBE-15.If using a Covaris instrument other than specified, contact Covaris.Handling the Library Card}}}8To avoid instrument damage, do not place the library card guide on the library cardduring library card verification or a run.Use the library card latch release to load and remove the library card from the librarycard stage.} Do not snap the library card into place.} Oil and reagents in a used library card can splash out of the card and onto theinstrument.Hold the used library card level when removing it from the instrument to avoidspilling its contents.Material # 20000943Document # 15049722 v01
}}}}}}}}}Load the library card while it is on the library card stage to avoid spilling ordisturbing the loaded contents.The NeoPrep Control Software guides you through the steps to set up a run and loadthe library card. Use the loading procedures in this guide as a reference.Do not open the compartment door during library card verification or a run.Change your gloves after loading the oil.Transfer contents from the reagent plate to the corresponding wells on the librarycard.Reference the corresponding colors and well labels on the reagent plate and librarycard guides.Use the pipettes and tips specified in Pipette and Tip Requirements on page 7 andConsumables and Equipment on page 31. Other pipettes and tips are not supported andcan result in reagents not dispensing properly and run failure.Use a multichannel pipette to load reagents, samples, and adapters.To avoid instrument damage, make sure that the library card guide is removed fromthe library card before starting the run.Library Card Loading Techniques}}}}}}}Use proper library card loading techniques and specified loading angles.Pipette to the first stop to avoid creating bubbles.Insert pipette tips perpendicular to the well.Insert the pipette tips to the bottom of the well while dispensing. Do not lift the tipsuntil the reagents are dispensed completely.Dispense at an angle by pointing pipette tips under the well label and dotted welloutline on the library card guide.The pipette loading angle depends on the item being dispensed. The angle isspecified in each step of the control software loading guide and is depicted in theprotocol.An icon represents the loading angle and the volume is specified on the controlsoftware loading guide. For example,IconDescriptionPoint the pipette tip toward the well label and dotted well outline on the left.Point the pipette tip perpendicular in the well.Point the pipette tip toward the well label and dotted well outline on the right.}Increase the pipette angle if liquid is not dispensing from the pipette tips.Handling Samples}}}}}Always track the location of each sample.Change tips between each sample to avoid cross-contamination.Do not centrifuge samples before loading.Some space might be present in the pipette tips during transfer from the sampleplate to the library card.Both 350 bp and 550 bp sample insert sizes can be included in a single run.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide9Tips and TechniquesLibrary Card Loading Guidelines
Collecting Libraries}}}}}}}Unload the library card while it is on the library card stage.The NeoPrep Control Software guides you through the steps to unload the librarycard. Use the procedures in this guide as a reference.Do not use a 20 µl pipette. It does not fit properly into the library card well.Insert pipette tips perpendicularly and touch the tips to the bottom of the collectionwells.Hold down the library card with one hand while removing the tips from thecollection ports to prevent any movement of the card.An icon represents the required pipette angle, and the volume is specified on thecontrol software unloading guide. For example,Inspect each pipette tip to make sure that a blue library droplet is present in the tipsindicated by the control software.Figure 1 Library Droplet in Pipette Tips}}If a blue library droplet is not visible in each expected pipette tip, do the following:} Transfer the extracted liquid to the corresponding plate well containing RSB.} Do not dispense the liquid back into the library card, which can introduce air gapsand interfere with library extraction.} Use a single-channel pipette to repeat the transfer 1 time for the wells that did notcontain the blue droplet. Do not attempt the transfer more than 2 times.Vigorously pipette up and down in RSB to dislodge the blue library droplet from thepipette tip.Handling Library Separation Tube Strips}}}10Label the tubes to support tracking sample location.Use the wells of a plate or another device to hold the library separation tube stripsupright.Do not centrifuge library separation tube strips.Material # 20000943Document # 15049722 v01
Library Prep WorkflowLibrary Prep WorkflowFigure 2Workflow DiagramSamples are fragmented manually using a Covaris Focused-ultrasonicator. Then, thesamples are size-selected and concentrated using Digital Microfluidics Beads (DMB) anda Sample Concentration Solution buffer (SC350 or SC550). Oil, samples, reagents, andadapters are loaded into the library card for a run on the NeoPrep System. A runincludes library prep, and optional quantification, and normalization. The NeoPrepControl Software guides you through the run setup and library card loading steps. AfterTruSeq Nano DNA Library Prep for NeoPrep Reference Guide11
the run is complete, the control software guides you through the process of collectingyour libraries from the library card and separating them from the oil. If quantificationand normalization were performed by the NeoPrep System, the libraries are ready forpooling, denaturing, and clustering.Before proceeding:} Review Best Practices, available on the Illumina website. See Additional Resources onpage 5.} Review Supporting Information on page 30. Confirm kit contents and make sure thatyou have the requisite equipment and consumables for this protocol.12Material # 20000943Document # 15049722 v01
Before beginning library preparation, plan for your NeoPrep System run.1Select the samples to use for library prep. Each kit is single-use for 1 NeoPrep Systemrun and each NeoPrep System run prepares up to 16 samples.2Plan the sample locations on the sample plate and the library card.} Place samples 1–8 in column 1, A–H} Place samples 9–16 in column 2, A–H3Use the default index adapters in the order that they are arranged in the reagentplate and arrange the samples used with those index adapters accordingly.Figure 3 TruSeq Nano DNA Library Prep for NeoPrep Index AdaptersABCAdapters A–H (default for samples 1–8)Adapters I–P (default for samples 9–16)Adapters Q–X (alternate)} Indexes are single-use.} Each sample requires a unique index in a library prep run.} Each kit includes 24 single-index adapters, allowing for pooling up to 24 sampleswith multiple NeoPrep System runs.} For the index adapter layout, see Reagent Plate Contents on page 30.} For the index adapter sequences, see Index Adapter Sequences on page 34. TheTruSeq Nano DNA Library Prep for NeoPrep adapters are TruSeq LT single-indexadapters.} Review the planning steps in the TruSeq Library Prep Pooling Guide (document #15042173) for Illumina sequencing systems that require balanced indexcombinations.4[Optional] Use IEM or BaseSpace Prep tab to record information about your samplesand indexes. The information is used during the run setup.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide13Select Samples and IndexesSelect Samples and Indexes
Fragment DNAThis process describes how to shear input gDNA into 350 bp and 550 bp dsDNAfragments required for loading into the library card.Consumables}}}}}RSB (Resuspension Buffer)96-well 0.3 ml PCR plates (2)Covaris microTUBE-15 AFA Beads Screw-Cap (1 per sample)gDNA samples} 25 ng for 350 bp inserts} 75 ng for 550 bp insertsMicroseal 'B' adhesive sealPreparation1Remove RSB from 2 C to 8 C storage.2Turn on the Covaris instrument and follow manufacturer instructions to set up yourinstrument.3Make sure that pipettes are calibrated before beginning. Uncalibrated pipettes canlead to variations in insert size, and reagents not dispensing properly, resulting inrun failure.Procedure1Quantify gDNA samples using a fluorometric-based method that uses dsDNAbinding dyes such as Qubit or QuantiFlour.2Normalize gDNA samples with RSB in a final volume of 15 µl in separate wells of anew PCR plate:} For a 350 bp insert size—1.67 ng/µl of sample} For a 550 bp insert size—5 ng/µl of sample3Transfer 15 µl of each normalized DNA sample to a separate microTUBE-15.4Centrifuge, using a microTUBE adapter, at 3000 g for 1 minute.5Fragment the DNA on a Covaris using the appropriate settings for your instrument.Table 2 Covaris S220 or E220 SettingsSettingDuty factorPeak Incident PowerCycles per burstDurationTemperatureWater Level—S220Water Level—E22014350 bp Insert550 bp Insert20%18 W5045 seconds22 seconds20 C1510Material # 20000943Document # 15049722 v01
Fragment DNATable 3 Covaris M220 SettingsSettingDuty factorPeak Incident PowerCycles per burstDurationTemperature350 bp Insert550 bp Insert20%30 W5042 seconds23 seconds20 C6Centrifuge, using a microTUBE adapter, at 600 g for 5 seconds.7Transfer 15 µl fragmented DNA from each microTUBE-15 to a separate well of a newPCR plate.SAFE STOPPING POINTIf you are stopping, seal the plate and store at -25 C to -15 C for up to 7 days.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide15
Prepare Samples for LoadingThis process describes how to perform size-selection using SC350 or SC550. Both 350 bpand 550 bp insert sizes can be included in a single run. DMB binds the DNA inpreparation for loading onto the library card.Consumables}}}}}}}DMB (Digital Microfluidics Beads)SC350 (Sample Concentration Solution 350) for 350 bp insertsSC550 (Sample Concentration Solution 550) for 550 bp inserts1.5 ml microcentrifuge tubes (1 per library insert size)96-well 0.3 ml PCR plateMicroseal 'B' adhesive sealRNase/DNase-free reagent reservoirs (2)About ReagentsSC350 and SC550} When running both 350 bp and 550 bp insert sizes on the same library card, use aseparate, new 1.5 ml microcentrifuge tube for each solution.} When creating separate mixtures for both 350 bp and 550 bp insert sizes, add theDMB volume specified to each tube containing SC.} If you are creating separate mixtures for both 350 bp and 550 bp insert sizes, poureach mixture into a separate, new reagent reservoir.} Make sure that the mixture added to the sample plate well contains the SC thatcorresponds to the desired library insert size.Preparation1If the sample plate was stored, thaw it at room temperature, and then centrifuge at280 g for 1 minute.2Prepare the following consumables:ItemDMBSC350SC550Storage2 C to 8 C2 C to 8 C2 C to 8 CInstructionsLet stand for 10 minutes to bring to room temperature.Let stand for 10 minutes to bring to room temperature.Let stand for 10 minutes to bring to room temperature.Procedure161Add 1 of the following to a new 1.5 ml microcentrifuge tube:} For a 350 bp insert size—900 µl SC350} For a 550 bp insert size—700 µl SC5502Vortex DMB until well-dispersed. Do not centrifuge DMB.3Add 100 µl DMB to the microcentrifuge tube containing SC350 or SC550. Vortex for5 seconds.4Pour the DMB and SC mixture into a new reagent reservoir.Material # 20000943Document # 15049722 v01
Add 35 µl DMB and SC mixture to each well of the sample plate. Pipette to mix.6Shake or vortex at 1400 rpm for 12 minutes.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide17Prepare Samples for Loading5
Set Up Run and Load Library CardThis process describes how to set up a NeoPrep System run, which includes loading oil,samples, reagents, and adapters into the library card. Load the library card and start therun within 90 minutes.The NeoPrep Control Software guides you through the steps to set up a run and load thelibrary card. Use the procedures in this section as a reference. For more information onthe NeoPrep Control Software, see the NeoPrep Library Prep System Guide (document #15049720).Consumables}}}}}}}Library cardLibrary card guideOil vialOil funnelDMB (Digital Microfluidics Beads)TruSeq Nano DNA - NeoPrep reagent plate[Optional] RSB (Resuspension Buffer)WARNINGThe reagent plate contains hazardous materials. Personal injury can occur throughinhalation, ingestion, skin contact, and eye contact. Wear protective equipment, includingeye protection, gloves, and a laboratory coat. Handle the used reagent plate as chemicalwaste. Dispose of containers and any unused contents in accordance with thegovernmental safety standards for your region. For more information, see the SDS forthis kit at support.illumina.com/sds.html.Preparation1Prepare the following consumables:ItemStorageInstructionsReagent plate -25 C to -15 C Let stand for 15 minutes to bring to room temperature.RSB2 C to 8 CIf stored, let stand for 10 minutes to bring to roomtemperature.Set Up the Run181Vortex the reagent plate for 3 seconds.2Centrifuge at 600 g for 5 seconds.If you are not using the reagent plate immediately, set aside on ice.3Select Prepare Libraries on the NeoPrep System Welcome screen.4Do the following and then select Next.} If running in BaseSpace mode, select a run.} If running in standalone mode, use the following options to select a protocol:} Select Select by barcode, and then scan the reagent plate barcode or enter thereagent plate serial number.} Select Select by name, and then select TruSeq Nano DNA.Material # 20000943Document # 15049722 v01
Configure the run. Select Next.For more information, see Configure the Run in the NeoPrep Library Prep System Guide(document # 15049720).} The following processes are available:} Prep Library—Prepares libraries and must be selected.} [Optional] Quantify—Quantifies samples during the run, after library prep iscomplete.} [Optional] Normalize—Normalizes the final libraries to 10 nM, afterquantification is complete. This option can only be selected if Quantify is alsoselected.} Configuration options for TruSeq Nano DNA are as follows:Table 4 Configuration OptionsParameterSample CountPCR CyclesInsert Size (bp)DefaultOptions161–1664–15No default 350, 550, MixedNOTEOnly the default PCR Cycles setting is supported.} Select the Insert Size. If the Mixed Insert Size option is selected, the insert sizefor each sample must be specified when confirming the run settings in step 6.6Review the run and sample information. Select Next.For more information, see Confirm Run in the NeoPrep Library Prep System Guide(document # 15049720).If the Mixed Insert Size option was selected for the run configuration, the insert sizemust be selected for each sample in the Sample Info tab.7Enter the consumable tracking information. Select Next.For more information, see Track Consumables in the NeoPrep Library Prep System Guide(document # 15049720).8Open the library card compartment door, slide the latch release to the right, and thenplace the library card on the library card stage.WARNINGTo avoid instrument damage, make sure that the library card guide is not on thelibrary card.9Close the library card compartment door. Select Verify Library Card. Do not openthe compartment door during library card verification.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide19Set Up Run and Load Library Card5
Load the Library Card1When library card verification is complete, open the library card compartment doorand place the library card guide on the library card.Figure 4 Reagent Plate to Library Card Transfer LayoutABCDEFG2Reagent plateLibrary cardOilLarge reagentsSmall reagentsSamplesAdaptersLoad the entire contents of the oil vial into the library card using the oil funnel. Wait3 minutes for the oil to drain. Change your gloves after loading the oil.WARNINGUse the pipette tips specified in Pipette and Tip Requirements on page 7 and Consumables andEquipment on page 31. Other tips are not supported and can result in reagents notdispensing properly and run failure.The loading angle of the pipette depends on the item being dispensed. The angle isspecified in each step of the control software loading guide and is depicted in theseprocedures.3Transfer 45 µl of prepared samples 1–8.Figure 5 Loading Samples 1–820Material # 20000943Document # 15049722 v01
Transfer 45 µl of prepared samples 9–16.Figure 6 Loading Samples 9–165If you are preparing 16 samples, add 45 µl RSB to empty sample wells.NOTEIf you are preparing 9 samples, the NeoPrep Control Software does not provide theRSB loading instructions for wells 9–16. Add 45 µl RSB to each empty sample well 9–16.6Transfer 125 µl of the large reagents i–iv.Figure 7 Loading Large Reagents i–iv7Transfer 125 µl of the large reagents v–vii.Figure 8 Loading Large Reagents v–viiTruSeq Nano DNA Library Prep for NeoPrep Reference Guide21Set Up Run and Load Library Card4
8Vortex DMB until well-dispersed. Do not centrifuge DMB.9Add 60 µl DMB to the large reagent well viii.Figure 9 Loading Large Reagent viii10 Transfer 15 µl of small reagents 1–4, and then 5–8.Figure 10 Loading Small Reagents 1–4, 5–811 Transfer 5 µl of small reagents a–d, and then e–h.Figure 11 Loading Small Reagents a–d, e–hNOTEFor steps 12 and 13, if you are not using the default index adapter layout, the controlsoftware specifies which adapter to transfer to each library card well.12 Transfer 3 µl of adapters A–H.Figure 12 Loading Adapters A–H22Material # 20000943Document # 15049722 v01
Figure 13 Loading Adapters I–PNOTEIf you are preparing 9 samples, the NeoPrep Control Software does not provide theadapter loading instructions for adapters I–P. Transfer 3 µl of adapters I–P.14 Remove the library card guide. Keep it for later use during the unloading process.WARNINGTo avoid instrument damage, make sure that the library card guide is removed fromthe library card.15 Close the library card compartment door. Select Start Run. Do not open thecompartment door until the run is complete.16 When the run is complete, select Next. Libraries can remain at room temperature ona library card for up to 3 days after a run is complete.TruSeq Nano DNA Library Prep for NeoPrep Reference Guide23Set Up Run and Load Library Card13 Transfer 3 µl of adapters I–P.
Unload LibrariesThis process describes how to collect libraries from the library card, separate librariesfrom the oil, and unload the library card from the instrument.The NeoPrep Control Software guides you through the steps to unload the library card.Use the procedures in this section as a reference. For more information on the NeoPrepControl Software, see the NeoPrep Library Prep System Guide (document # 15049720).Consumables}}}}}RSB (Resuspension Buffer)Library card guideLibrary separation tube strips (2)96-well 0.3 ml PCR plates (2)Microseal 'B' adhesive sealsWARNINGThe used library card contains hazardous materials. Personal injury can occur throughinhalation, ingestion, skin contact, and eye contact. Wear protective equipment, includingeye protection, gloves, and a laboratory coat. Handle the used library card as chemicalwaste. Dispose of containers and any unused contents in accordance with thegovernmental safety standards for your region. For more information, see the SDS forthis kit at support.illumina.com/sds.html.Preparation1Remove RSB from 2 C to 8 C storage and bring to room temperature.2Label wells of 2 new PCR plates 1–16.3Label tubes of a library separation tube strip 1–8 and another library separation tubestrip 9–16.Procedure241Add 10 µl RSB to each well of a new PCR plate labeled 1–16.2Open the library card compartment door and place the library card guide on thelibrary card.Material # 20000943Document # 15049722 v01
Use a 200 µl pipette to transfer 20 µl from library card collection wells 1L–8L, andthen 9L–16L to corresponding wells 1–16 of
ILLUMINAPROPRIETARY Catalog#NP-101-9001DOC Material#20000943 Document#15049722 v01 October2015 TruSeq NanoDNA LibraryPrepforNeoPrep ReferenceGuide .
