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ii Part# 15018210 Rev. D Revision History

Part # Revision Date Description of Change 15018210 D April 2012 • Corrected ratio of MCS4 and RTE reagent tubes per sample in the Pre- Qualify cDNA Samples procedure's consumables table. • Removed WG-DASL HT Assay Guide catalog number. 15018210 C December 2011 • MCS3 reagent replaced with MCS4 and RTE and the protocol revised. • DASL Single-Use cDNA Synthesis Kit renamed to cDNA Synth MCS4 Single- Use Kit. • AM1 Washes - Added step to place ASE plate on magnetic plate. 15018210 B December 2010 Specify Image Beadchip scan settings. 15018210 A December 2010 Initial release

Whole-Genome Gene Expression DASL HT Assay Guide iii iv Part# 15018210 Rev. D Table of Contents

Revision History iii Table of Contents v List of Tables vii

Chapter 1 Overview 1 Introduction 2 WG-DASL HT Assay 3 RNA Samples 4 WG-DASL HT Assay Workflow 6 BeadChips 11 HiScan, iScan, BeadArray Reader, AutoLoader and AutoLoader2 12 GenomeStudio 13 Illumina Lab Protocols 14

Chapter 2 Standard Operating Procedures 15 Introduction 16 Acronyms 17 Lab Setup 20 Lab Maintenance 22 Safety Precautions 24 Best Practices 25 RNase-Free Techniques 28 Standard Lab Procedures 29 Tracking Tools 31 Equipment, Materials, and Reagents 34

Chapter 3 WG-DASL HT Assay Lab Protocols 43 Introduction 44 Quantitate RNA (Optional) 45

Whole-Genome Gene Expression DASL HT Assay Guide v Pre-Qualify cDNA Samples (Optional) 56 Make Single-Use RNA (SUR) Plate 60 Make Assay-Specific Extension (ASE) Plate 63 Add Master Mix for Extension & Ligation (MEL) 66 Make PCR Plate 70 Inoculate PCR Plate 72 Thermal Cycle PCR Plate 76 Bind PCR Products 77 Make Intermediate (INT) Plate 80 Precipitate and Wash INT Plate 83 Hybridize BeadChip 87 Wash BeadChip 95 Image BeadChip 111 GenomeStudio 112

Appendix A WG-DASL HT AssayControls 113 Introduction 114 View the Control Report 115 Negative Controls 116 Oligo Annealing Controls 117 Array Hybridization Controls 118 Gene Intensity (Housekeeping and All Genes) 119

Appendix B Assay Qualification Using gDNA 121 Introduction 122 Make Single-Use DNA (SUD) Plate 123 Data Analysis 126

Index 127

Technical Assistance 131

vi Part# 15018210 Rev. D List of Tables

Table 1 AutoLoader and AutoLoader2 Features 12 Table 2 WG-DASL HT AssayAcronyms 17 Table 3 Vortexer Calibration Speeds 29 Table 4 Sample Sheet Guidelines 31 Table 5 Illumina Equipment Options for the WG-DASL HT Assay 34 Table 6 User-Supplied Equipment 35 Table 7 User-Supplied Materials 36 Table 8 WG-DASLHT Assay Kits 38 Table 9 WG-DASL HT Assay Kit Contents 39 Table 10 User-Supplied Reagents 40 Table 11 Concentrations of Standard Ribosomal RNA 47 Table 12 Volumes for RiboGreen Reagents 49 Table 13 Primers for qPCR using SYBR Green Detection 56 Table 14 Thermal Cycler Program 76 Table 15 WG-DASL HT Assay Scan Settings 111 Table 16 Illumina General Contact Information 131 Table 17 Illumina Customer Support Telephone Numbers 131

Whole-Genome Gene Expression DASL HT Assay Guide vii viii Part# 15018210 Rev. D Chapter 1 2 3 4 6 1 11 12 13 14 Chapter 1 Overview HiScan, iScan, BeadArray Reader, AutoLoader and AutoLoader2 GenomeStudio Illumina Lab Protocols Introduction Overview WG-DASL HT Assay RNA Samples WG-DASL HT Assay Workflow BeadChips Whole-Genome Gene Expression DASL HT Assay Guide Overview 2 Introduction TAsyofr aallaayi fu o1 ape toc,adi dpal ohigh to adaptable is offers: and Assay once, HT at WG-DASL The samples 12 to up of automation. WG-DASLthroughput analysis The parallel tissues. offers paraffin-embedded degradation, formalin-fixed, Assay from partial HT undergone derived RNAs have that as such samples enabling RNA methodologies in unique measurements incorporates expression Assay HT WG-DASL The savings. 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Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Overview 4 eomne uiiainKit Purification Recommended RNAs Degraded Partially Samples RNA 4 3 steps: 2 these includes Kit Paraffin RNA Pure 1 High the using purification RNA brief, In from Kit the Paraffin RNA in Pure RNA Science. High the Applied of the Roche performance recommends the Illumina and substantially Assay. degradation HT tissues RNA WG-DASL FFPE of from degree derived the RNA both of impacts purification for used method The intact of abundance reduced the for compensate to ng/µl, FFPE-derived 200 sequences. with target to best performs 40 Assay from HT ranging (FFPE) WG-DASL partially embedded RNAs the paraffin in RNAs, formalin-fixed, expression these from gene For derived monitor tissues. those to as used be such also RNAs, degraded can Assay HT WG-DASL The assay optimal to conducive input not RNA are of performance. and levels Lower replicates, total among 20–100 ng/µl. reproducibility intact from decrease accept by ranging followed can concentrations method, Assay in standard HT RNA WG-DASL RNAs any The The using formamide. water. 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Assay HT WG-DASL Illumina overall the describes section This iue1 Figure GDS TAsyWorkflow Assay HT WG-DASL at#1081 e.D Rev. 15018210 # Part WG-DASL HT Assay Workflow 7 . on page 60 Hybridize cDNA to Assay Oligonucleotides Reverse Transcribe RNA with SUR enzyme Figure 3 Figure 2 Make Single-Use RNA (SUR) Plate The WG-DASL HT Assay monitorschimeric-oligos gene containing expression universal by targeting PCR sequencesoligos amplification with (ASOs) primer are sites. used Two to assay-specificdownstream-specific assay oligo each (DSO), each transcript: of anand which upstream-specific a contains oligo gene-specific (USO) a sequence and universal thatthe a complements PCR BeadChip. a priming site corresponding capture sequence on 29,285 unique assay probesthe are National used, Center corresponding for to Biotechnology up-to-date Informationdatabase content Reference derived (Release Sequence from 38 (NCBI RefSeq) (Nov 7, 2009). 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The This BeadChip built-in manufacturingbased redundancy process quality improves includes control robustness of hybridization- and eachquality, array reproducible arrays. feature, allowing consistent production of high- Whole-Genome Gene Expression DASL HT Assay Guide BeadChips Overview 12 AutoLoader2 and AutoLoader Reader, BeadArray iScan, HiScan, mgn ytmsta,s hti a cnteBaCis etrsinclude: Features BeadChips. the 1 Table scan can the it that in carriers so tray, BeadChips system’s placing respectively. by imaging AutoLoader BeadArray processing or or AutoLoader2 unattended System support optional AutoLoaders iScan the Both or with automated HiScan be the into can Reader BeadChips of unloading and assay. 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GenomeStudioexpression be exported analysis Gene and programs. analyzed You byon can most groups perform standard of these gene arrays analyses treated on as individualData replicates. analysis arrays or features of the GenomeStudio Gene Expression Module include: For feature descriptions and instructionsvisualize on and using analyze the GenomeStudio miRNAGenomeStudio platform data, to see Gene the Expression Module User Guide Whole-Genome Gene Expression DASL HT Assay Guide GenomeStudio Overview 14 luiaLbProtocols Lab Illumina o ntutoso o opromteW-ALH sa rtclo edhp,see BeadChips, Protocols on Lab protocol Assay Assay HT WG-DASL HT WG-DASL the 3 perform up Chapter to set to how how on instructions areas. explains For post-PCR and and lab pre- assay separate Illumina maintain an and for of tools risk and the procedures minimize operating and efficiency promote to contamination. designed are protocols lab Illumina hpe tnadOeaigProcedures Operating Standard 2 Chapter . ecie h standard the describes at#1081 e.D Rev. 15018210 # Part Chapter 2 16 17 20 22 24 25 28 29 31 34 15 Chapter 2 Standard Operating Procedures Acronyms Lab Setup Lab Maintenance Safety Precautions Best Practices RNase-Free Techniques Standard Lab Procedures Tracking Tools Equipment, Materials, and Reagents Introduction Standard Operating Procedures Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 16 Introduction bandalo h eust qimn,mtras n reagents. have with and and familiar materials, recommendations, equipment, are the requisite you the all that of implemented assume all have guide obtained chapter, this this of of rest contents the the in and described protocols materials, equipment, assay standard The of lists find an also operating will reagents. for You precautions lab. and assay procedures operating Illumina standard explains chapter This at#1081 e.D Rev. 15018210 # Part Acronyms 17 Definition Assay-Specific Extension Plate Assay Specific Oligo Complementary DNA DASL Assay Pool cDNA Mediated Annealing, Selection, Extension, and Ligation Diethyl Pyrocarbonate Deoxyribonucleic Acid Downstream-Specific Oligo Wash Buffer Ethanol Humidity Control Buffer High Throughput High Temperature Wash Buffer Hybridize or Hybridization Hybridization Buffer Intermediate Plate Add MEL 1 Reagent HT INT ASE ASO DSO Hyb HYB DAP HCB AM1 DNA HTW E1BC EtOH DASL DEPC cDNA WG-DASL HT AssayAcronyms Acronym Table 2 Whole-Genome Gene Expression DASL HT Assay Guide Acronyms Standard Operating Procedures 18 Acronym RNase QRNA NaOH MCS4 PMPs MH1 RNA SCM MPB MEL SUR PCR UB2 UB1 SDS RTE OB1 PS1 PB1 IP1 aeHb1Reagent 1 Hyb Make Reagent Extension/Ligation for Mix Master 4 Use Single for Synthesis cDNA Mix Master Reagent 1 PCR Inoc nvra ufr2Reagent 2 Buffer Universal Reagent 1 Buffer Universal Plate RNA Single-Use Sulfate Dodecyl Sodium Mix Master Color Single Enzyme Transcriptase Reverse Ribonuclease Acid Ribonucleic Quantification RNA Reagent Solution Precipitation Particles Paramagnetic Plate Reaction Chain Polymerase hybridization for BeadChips prepare to used Reagent Reagent 1 Buffer Binding DNA & Hybridization Oligo Hydroxide Sodium Reagent B Particle Magnetic Definition at#1081 e.D Rev. 15018210 # Part Acronyms 19 Definition Multiple of Gravitational Acceleration Uracil DNA Glycosylase Upstream-Specific Oligo Whole-Genome Gene Expression DASL XStain BeadChip Solution 4 xg USO XC4 UDG Acronym WG-DASL Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 20 eiae qimn n Supplies and Equipment Dedicated Areas Post-PCR and Pre- Separate Setup Lab olwteerlst vi otmntn h r-C area: pre-PCR the contaminating avoid areas. to between rules instruments these the the Follow to share etc.) block, Never heat areas. post-PCR oven, centrifuges, and (pipettes, pre-PCR instruments of sets separate Dedicate space. laboratory dedicated separate, a in processes pre-PCR the perform of possible, resumption If delay significantly and processes product operations. lab PCR normal down results. unreliable shut and can reagents, inaccurate contamination contaminate causing may samples, products and PCR exercised, instrumentation, is caution sufficient Unless Contamination Product PCR Prevent processes). 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Provide with training a for 0.5% personnel sodium responsiblehow for to cleaning prevent the PCR lab product areas contamination. so that they know Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 24 References Precautions Safety laevstht:/w.luiacmmd osetelts aeildt aeysheets. safety data material latest the see to http://www.illumina.com/msds visit Please laerfrt oenetladfclt aeysadrsapial oyu site. your to applicable standards safety facility and governmental to refer Please CAUTION which kit, this for MSDS the see http://www.illumina.com/msds. information, at available more is standards For safety region. your governmental any for the and with containers accordance of in Dispose contents contact. unused eye and inhalation, probable contact, through a skin occur is ingestion, can that injury amide aliphatic Personal an toxin. of reproductive use the involves protocol This WARNING post-amp and pre-amp samples. among cross-contamination biological avoid handling to qualified when samples caution by Exercise performed only. be personnel should guide laboratory this in described protocols The CAUTION at#1081 e.D Rev. 15018210 # Part Best Practices 25 CAUTION To prevent evaporation and spills,and which cross-contamination, could lead ensure to that assay allthe variability 96 wells. caps are securely seated in To optimize your data andwhenever minimize performing the errors WG-DASL and HT waste, Assay read protocols. and follow best practices The floor is contaminated withcoming PCR from product the transferred post-PCR on area.contaminated. the Therefore, treat shoes anything of falling individuals to the floorDisposable as items if falling it to were thecoat ground, hangers such should as be empty thrownassay. tubes, away During pipette at tips, the the assay, gloves, end or never of lab touchNon-disposable the any items day items falling or that at to have the thecontainers, ground, completion fallen should such of to be the as the immediately ground. pipettes andhypochlorite or (10% thoroughly important cleaned bleach) sample with solution a to remove 0.5%Use product sodium contamination. a 0.5% sodiumcontacted hypochlorite (10% the contaminated bleach) solution item. to cleanIndividuals any handling lab anything surface that thatthrow has has away fallen their to lab the gloves floor, and disposable put or on not, a must new pair. As a convention, apply barcode labels to the right side of plate (column #12 end). Never reuse excess reagents. Discard according to your facility requirements. Orient the cap mat so that A1 on the cap matches A1 on the plate. Whole-Genome Gene Expression DASL HT Assay Guide Best Practices Items Falling to the Floor Applying Barcode Labels to Plates Reagent Reuse Handling Cap Mats Standard Operating Procedures 26 rclDAGyoyae&dUTP & Glycosylase DNA Uracil 96 Samples than Fewer Preparing Handling BeadChip Carefully Pipette rtcl hnyupaetecpmtbc ntepae esr omthi oits to it match to the sure in be later plate, correctly. use the it for on orient location back and the safe mat plate a splashing cap original in the avoid down, to place upside slowly you aside, When and protocol. carefully mat so cap do the Set mat, contents. cap a remove you When h C atrmxcnan aacdmxueo h olwn items: following the of mixture balanced a contains mix master PCR Assay The HT WG-DASL prevent the help purchase to can mix You master PCR the to (UDG) contamination. 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RNases human the from most on contamination found prevent to experiments throughout gloves Wear at#1081 e.D Rev. 15018210 # Part Standard Lab Procedures 29 xx-xx-xx xx-xx-xx xx-xx-xx xx-xx-xx 1800 rpm 2000 rpm 2200 rpm 1600 rpm 1800 rpm 1625 rpm 1975 rpm 1450 rpm Vortexer Calibration Speeds Display Speed Actual Vortex Speed Calibration Date Table 3 Set the digital stroboscope display speed toTurn 1600 the rpm. vortexer on and1600 adjust rpm. the vortexer speed until the actualRecord the speed displayed reaches vortexer speed andspeed of note 1600 down that rpm. it represents anUse actual the same method describedactual above vortex to speeds determine the of displayed 1,800,the speed 2,000, WG-DASL for HT and the Assay. 2,200 rpm. These vortexPlace speeds are a used label in on theexample vortexer of with a the vortexer calibration calibration information. label The you following can is create an and affix to your vortexer. Running the WG-DASL HT Assayand protocols familiarize requires that yourself with you perform standard some procedures. basic setup Calibration The vortexer’s displayed speed mayrecommends vary using from a the digital actualhave stroboscope vortex to determined speed. determine the Illumina the actual actual vortexuse vortex speed, these speed. record Once measurements it you for along reference throughout with the the assay. displayed1 speed and 2 3 4 5 Whole-Genome Gene Expression DASL HT Assay Guide Standard Lab Procedures Calibrating and Using the Vortexer Standard Operating Procedures 30 laigadClbaigPipettes Calibrating and Cleaning Centrifuge the Balancing 1 Security for Straps Velcro Using s ut-hne iet odses reagents. dispense possible, Where to pipette decontaminated. multi-channel and clean, a calibrated, use properly are pipettes that Ensure possible. as being similar plate as each be opposite plate should weights balance The a centrifuged. place plates, centrifuge you Whenever iue8 Figure b Velcro three with follows: plate, the as a secure plates, to 96-well used securing is for which straps tray, top vortexer’s the Replace u he 0ic egh fVlr op.Ueteea tast eueplates secure to straps as these platform. Use vortexer loops. the Velcro onto of lengths to hooks 20-inch tray. three bottom these Cut platform Attach hooks. shaker Velcro the backed of underside adhesive the of lengths 2-inch six Cut ecoSrp nVree Platform Vortexer on Straps Velcro hnvryuuetevree,scr h ltswt h ecostraps. Velcro the with plates the secure vortexer, the use you Whenever CAUTION at#1081 e.D Rev. 15018210 # Part Tracking Tools 31 O Required (R) Optional (O) or GenomeStudio Gene Expression Module – guides you through the protocols. – used to record information about your samples for later – allows you to map RNA samples from plate to plate, or from Description Name of the sample. Used onlyIf for not display user-specified, in the the GenomeStudio table. assign application will a default sample name,plate concatenating and the sample sample well names. Example: S12345 NOTE Lab Tracking Forms can behttp://www.illumina.com/documentation. downloaded via Sample Sheet Guidelines . Create your sample sheet according to the following guidelines: Name Column Heading Sample_ Table 4 Experienced User Card Lab Tracking Form plate to BeadChip. Record theof position each of DAPs reagent used in in theSample the plate protocol. Sheet wells, template and the barcode use in data analysis. } } } To effectively track your samplessheet. and The assay, sample Illumina sheet recommends willanalysis. you later For create be instructions a used on sample byUser data the Guide GenomeStudio analysis, application see for the data Illumina provides the following tools for sample tracking and guidance in the lab: Create a copy of theoperator lab ID, tracking start form and for stop eachwhich times, run. samples reagent Use lot are it numbers placed to andonline on track barcodes, or information which and printed such arrays. and to as This record filled form in can by be hand. filled out and saved Whole-Genome Gene Expression DASL HT Assay Guide Sample Sheet Tracking Tools Lab Tracking Form Standard Operating Procedures 32 h olwn sa xml fteSml he omt h lcrncsml sheet sample electronic The http://www.illumina.com/documentation. format. Sheet via downloaded Sample be the of can template example an is following The Sample_ Sample_ Sample_ Heading Pool_ID Position Sentrix_ Sentrix_ Column Group Notes Plate Well ID aetesml he ne n aeyuws;freape h user-defined the example, for wish; you name name. any experiment format. under file sheet (.csv) sample comma-delimited the a Save in choose. be you columns must of sheet sample number Your any choose. contain you may information sheet sample whatever contain Your may header sheet sample Your hybridized. is A1 sample Example: the which to section BeadChip The 1529221001 Example: ID. BeadChip XS0007005-DAP Example: DAP. the of Name 1 Group Example: name. and default a group it one assigns creates Sample_ GenomeStudio the missing, If is group. Group sample the of name User-specified XS0005623-SUR table. Example: the in display samples. for RNA only containing Used plate the for name User-specified A01 Example: RNA 96-well the in plate. sample specific the containing well The Description at#1081 e.D Rev. 15018210 # Part pinl()or (O) Optional eurd(R) Required O O O O R R Tracking Tools 33 Sample Sheet Figure 9 The Sentrix_Position name corresponds toformat the being *.idat analyzed file (BeadChip). naming conventions for the Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 34 Equipment Reagents and Materials, Equipment, orIlmn con ersnaieo h aetIlmn rdc ud aalbeat (available guide product Illumina consult latest options, the kit http://www.illumina.com/literature). Gene or and representative and configuration account GoldenGate current the Illumina on both your BeadArray details or or For Kit kits. Starter iScan Option Universal HiScan, Expression the a either either need and you System Reader Assay, HT WG-DASL the perform To Equipment Illumina-Supplied and pre- for WG- necessary, the as for required stocks, separate all areas. are maintain post-PCR section to Remember this Assay. in HT listed DASL reagents and materials, equipment, The al 5 Table eeEpeso IT rdc pinKit Option Product (IVT) Expression Gene and Kit Option Product GoldenGate following: the of both or, Kit Starter Universal (optional) AutoLoader2 System iScan or System HiScan Item luiaEupetOtosfrteW-ALH Assay HT WG-DASL the for Options Equipment Illumina E1110 (220V) SE-101-1004 (110V) SE-101-1003 (220V) SE-101-1002 (110V) SE-101-1001 (220V) SE-101-1007 (110V) SE-101-1006 SY-201-1002 Dual-Scanner, SY-201-1001 Single-Scanner, SY-101-1001 SY-103-1001 luiaCtlg# Catalog Illumina at#1081 e.D Rev. 15018210 # Part Equipment, Materials, and Reagents 35 catalog # ab-0384/110, Source Cole-Parmer, A-87700-06, General lab supplier General lab supplier Molecular Devices, Gemini XS or XPS, www.moleculardevices.com Matrix Tech Corp., www.matrixtechcorp.com ABgene, catalog # 0563, www.abgene.com General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier User-Supplied Equipment Item Protective gloves Serological pipettes (50 ml) Stroboscope Aerosol filter pipette tips Heat sealer (Combi Heat Sealing Unit) Heat sealer adapter plate (Combi Heat Sealing Unit adapter plate) Lab coats Microtiter plate centrifuges (2, capable4°C) of 8–3000 xg, Note: Make sure this is 8-3000 xg, not 8-3000 rpm Orbital shaker Safety glasses Stopwatch/timer 96-well thermal cycler with heated lid [Optional] Fluorometer) 8-channel precision pipettes (5 µl to 200 µl) Table 6 User-Supplied Equipment The following user-supplied equipment is required. Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 36 Materials al 7 Table required. are materials user-supplied following The uevortexer Tube optical combination Tachometer/stroboscope, andar(uha eoo Whoosh-Duster) Aerosol as (such air Canned foil Aluminum pads Absorbent plates 200 Fluotrac flat-bottom black, 96-well plates V-bottom 96-well array well 8x12 microplates, skirted 96-well Cycler Thermal Item Item srSple Materials User-Supplied eea a supplier lab General supplier lab General www.coleparmer.com A-87700-06, # catalog Cole-Parmer, www.coleparmer.com Source www.vwr.com 16650-027, # catalog International, VWR supplier lab General supplier lab General www.gbo.com 655076, # catalog Greiner, www.vwr.com 29444-102, # catalog International, VWR www.abgene.com AB-0800, # catalog ABgene, www.mjr.com MSP-9601, # catalog Research, MJ Source at#1081 e.D Rev. 15018210 # Part Equipment, Materials, and Reagents 37 Source Millipore, catalog # MAHV-N45 10/50, www.millipore.com ABgene, catalog # AB-0592, www.abgene.com Phenix Research Products, catalog # LMT-SEAL-EX, www.phenix.com ABgene, catalog # AB-0812, www.abgene.com MJ Research, catalog # MSA-5001, www.mjr.com MJ Research, catalog # MSF-1001, www.mjr.com Labcor Products, Inc., catalog # 730-001, www.labcorproducts.com VWA International, catalog # 21007-970, www.vwa.com ABgene, catalog # AB-0566, www.abgene.com VWR International, catalog # 29444-102, www.vwr.com Centrifuge Alignment Frames, Millipore, catalog # MACF09604, www.millipore.com Item Microseal “A” PCR plate-sealing film Non-sterile solution basins (55 ml) [Optional] Foil stripper Microplate clear adhesive film (2 mil sealplate adhesive film, non-sterile) Microplate heat seals (heat sealing, EZ peel, clearpolystyrene for polypropylene plates) & Microseal “F” film Costar* 96-well plates, polypropylene, non-sterile,lids, V-bottom without Filter plates Cap mats (for deep-well plates,non-autoclavable) polypropylene, pierceable, Filter plate adaptor Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 38 Reagents al 8 Table the see http://www.illumina.com/literature. information, ordering at For sheet account catalog. data product Illumina appropriate your Illumina exact latest For consult the kits. options, or kit Assay representative and HT WG-DASL configuration the current on in details consumables the describes section This Reagents Illumina-Supplied hroSa etsaigfi sheets foil Heat-sealing Thermo-Seal capacity) (100 ml containers plastic Sterile GDS r C4w/UDG MCS4 1 Pre WG-DASL MCS4 1 Pre WG-DASL w/UDG MCS4 1 Pre WG-DASL MCS4 1 Pre WG-DASL Tweezers reservoir) (quarter reservoirs Sterile Item GDS TAsyKits Assay HT WG-DASL i Name Kit DA-905-1096 DA-905-0096 DA-905-1024 DA-905-0024 aao With # Catalog UDG X X www.abgene.com AB-0559, # catalog ABgene, www.beckmancoulter.com 372790, # catalog Coulter, Beckman supplier lab General eea a supplier lab General Source ape esof Sets Samples 96 96 24 24 at#1081 e.D Rev. 15018210 # Part Beadchips 8 8 2 2 Equipment, Materials, and Reagents 39 Quantity 3.2 ml 15 ml 5 ml 500 ml 12 samples each with 29,285 assays per sample 2.8 ml 1.7 ml 3.8 ml 2.2 ml 3.5 ml 4.0 ml 288 µl 3.5 ml 30 ml 950 ml 32 µl — WG-DASL HT Assay Kit Contents Item Oligo Hybridization & DNA Binding Buffer 1 Reagent (OB1) Reagent used to prepare BeadChips for hybridization (PB1) Single Color Master Mix Reagent (SCM) Barcode labels for the QRNA, SUR, ASE, PCR, and INT platesHigh Temperature Wash Buffer (HTW) 3HumanHT-12 X v4 each BeadChips Humidity Control Buffer (HCB) Hybridization Buffer Reagent (HYB) Inoc PCR Reagent (IP1) Magnetic Particle B Reagent (MPB) Make Hyb 1 Reagent (MH1) Master Mix for Extension Ligation Reagent (MEL) Master Mix cDNA Synthesis for Single Use 4 (MCS4) Precipitation Solution Reagent (PS1) Reverse Transcriptase Enzyme (RTE) Add MEL 1 Reagent (AM1) BeadChip Solution (E1BC) WG-DASL HT Assay Pool (DAP) (Based on up-to-date content derivedDatabase from (Release the 38 NCBI (Nov RefSeq 7, 2009)) Table 9 Whole-Genome Gene Expression DASL HT Assay Guide Standard Operating Procedures 40 al 10 Table required. are reagents user-supplied following The Reagents User-Supplied rclDAGyoyae(UDG) Glycosylase DNA Uracil (UB1) Reagent Buffer 1 Universal Ttnu a N polymerase) DNA Taq (Titanium Polymerase DNA Illumina-recommended samples 576 or samples, 96 (for Kit Single-Use MCS4 Synth cDNA bleach) (10% hypochlorite sodium 0.5% (Ethanol) EtOH and100% 70% 2-propanol hydroxide) (sodium NaOH 0.1N (XC4) 4 Solution BeadChip XStain (UB2) Reagent Buffer 2 Universal Item Item r-ulf DASmls(Optional): Samples cDNA Pre-Qualify srSple Reagents User-Supplied www.clontech.com 639220, # catalog Clontech, DA-950-6020 # catalog Illumina, DA-950-6010 # catalog Illumina, supplier lab General supplier lab General supplier lab General www.sigmaaldrich.com S0899, # catalog Sigma-Aldrich, Source nalkits all in included not 300 ml 25 ml eosiue ihEtOH with reconstituted when 350 ml Quantity at#1081 e.D Rev. 15018210 # Part Equipment, Materials, and Reagents 41 Source Roche Applied Science, catalog # 03 270 289 001 Invitrogen, catalog # R- 11490, www.invitrogen.com Item Quant-iT RiboGreen RNA Assay Kit Illumina-recommended High Pure RNA Paraffin Kit Whole-Genome Gene Expression DASL HT Assay Guide 42 Part# 15018210 Rev. D Chapter 3 44 45 56 60 63 66 70 72 76 77 80 83 87 95 111 112 43 Chapter 3 WG-DASL HT Assay Lab Protocols Quantitate RNA (Optional) Pre-Qualify cDNA Samples (Optional) Make Single-Use RNA (SUR) Plate Make Assay-Specific Extension (ASE) Plate Add Master Mix for Extension & LigationMake (MEL) PCR Plate Inoculate PCR Plate Thermal Cycle PCR Plate Bind PCR Products Make Intermediate (INT) Plate Precipitate and Wash INT Plate Hybridize BeadChip Wash BeadChip Image BeadChip GenomeStudio Introduction WG-DASL HT Assay Lab Protocols Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 44 Introduction h ntutosi hscatrasm htyuhv led aiirzdyourself familiarized already have appropriately. you that assume chapter with this protocols in the instructions shown. down The order scale the in samples, fewer protocol preparing each are Perform preparing you accordingly. for If protocols laboratory BeadChips. post-PCR for and 24 samples pre- detailed provides chapter This hpe tnadOeaigProcedures Operating Standard 2 Chapter opeae9 ape tonce. reagent at enough samples provides 96 WG- kit 96 sample prepare the a to Kit, using Reagent are Profiling you Assay If HT DASL Kit. WG- Reagent Profiling 24 sample Assay a HT using DASL 24 samples preparing describe procedures These NOTE n aestu h a area lab the up set have and at#1081 e.D Rev. 15018210 # Part Quantitate RNA (Optional) 45 By User 2° to 8°C-80°C User Make Single-Use RNA Quantity Storage Supplied 1 Up to 96 samples . CAUTION RiboGreen is susceptible to chemicalinformation, contaminants. For see the more Invitrogen website (www.invitrogen.com). on page 60 Item Quant-iT RiboGreen RNA Assay Kit, containing RiboGreen quantitation reagent, 20X TE,Ribosomal and RNA Standard RNA sample plate Estimated Time Hands-on time: ~30 minutes Fluorometer read time: ~5 minutes per plate Consumables This process uses thethe RiboGreen RNA WG-DASL HT quantitation Assay. kit You to96 samples. quantitate can RNA quantitate If samples up you already for to know(SUR) six Plate the plates, concentration, each proceed to containing upIllumina to recommends the Quant-iT RiboGreensamples. RNA The Assay RiboGreen Kit assay to candirectly. quantitate Other RNA quantitate techniques small may pick RNAproteins. volumes, up Illumina contamination and recommends measures such using RNA as aspecific small quantification. fluorometer because Spectrophotometry molecules might fluorometry provides and also RNA- that measure are DNA too and high. yield values Whole-Genome Gene Expression DASL HT Assay Guide Quantitate RNA (Optional) WG-DASL HT Assay Lab Protocols 46 aeSadr N Plate RNA Standard Make 2 standard of 1. dilutions 1 column serial of with wells plate the RNA in Standard RNA a ribosomal create you process, this In Preparation } } } } } d 0µ iooa N owl A1. well labeled to plate RNA the ribosomal in 20 µl B1–H1 Add to 20X) at kit RiboGreen RNA”. in (supplied “Standard TE 1X 10 µl Add each for Sample_Plate and (optional) Sample_Name the Sample_Well. enter Sheet, Sample the the contain In will plate This QRNA.” RNA. “Sample quantitated plate Fluotrac QRNA.” other the “Standard Hand-label plates Fluotrac the of one RNA.”Hand-label mix. to “Standard vortex plate then microtiter the and temperature Hand-label room to reagents all Thaw 0 lo 5 lNleebottle Nalgene 250 ml or 100 ml plate flat-bottom 96-well 200 Fluotrac plate microtiter 0.65 ml 96-well Item kit RiboGreen per 1 plate RNA Sample per 1 plate RNA Std per 1 samples 96 per 1 uniySoaeSupplied Storage Quantity instructions manufacturer’s See at#1081 e.D Rev. 15018210 # Part User User User By Quantitate RNA (Optional) 47 Do not transfer 10 10 10 10 10 10 20 10 (µl) Final Volume in Well 0 50 25 100 12.5 6.25 3.125 1.5262 (ng/µl) Concentration Dilution of Ribosomal RNA Standard Concentrations of Standard Ribosomal RNA B1 E1 F1 A1 C1 D1 G1 H1 Row- Column Transfer 10 µl from well A1 to wellChange B1. tips. Pipette Transfer up 10 µl and fromtimes. down well several B1 times. to well C1. PipetteRepeat up for and wells down C1, several D1,from E1, well F1, G1 and to G1, H1. changing tips eachTable time. 11 Figure 10 4 5 3 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 48 iueRiboGreen Dilute lts omk h N loec hnra ihtefluorometer. the QRNA with Sample read 1 and when QRNA fluoresce Standard RNA the the both make to to added plates, be will RiboGreen diluted The 7 6 iue11 Figure utpe9-elQN lts o ee hn9 N ape,saedw the down scale samples, 96 RNA than for fewer volumes. reagent For diluted plates. produce 1X QRNA to needed plates. 96-well 43 ml multiple volumes 6 and the to RiboGreen identifies up table 215 µl on following plate, so The 1 and for plates, TE 2 1X for foil. a TE 23 ml aluminum and and in RiboGreen supplies wrapped kit bottle 115 µl the Nalgene Use using 250 ml TE, or 1X into 100 ml RiboGreen sealed of dilution 1:200 a Prepare seal. to adhesive Proceed an with plate RNA Standard the Cover eilDltoso iooa RNA Ribosomal of Dilutions Serial iueRiboGreen Dilute npg 48 page on . at#1081 e.D Rev. 15018210 # Part Quantitate RNA (Optional) 49 23 43 63 83 123 (ml) 1X TE Volume 115 215 315 415 615 (µl) RiboGreen Volume Volumes for RiboGreen Reagents 1 2 3 4 6 Plates # QRNA Cap the foil-wrapped bottle and vortex to mix. Pour the RiboGreen/1X TE dilution into aUsing clean a reagent multichannel reservoir. pipette, transferwell 195 µl of columns RiboGreen/1X TE 1 dilution and into 2 each Add of 2 µl the Fluotrac of plate each labelled standardcolumns “Standard ribosomal 1 QRNA“. RNA and dilution 2 from of the the Standard Standard RNA QRNA plate Fluotrac to plate. Table 12 2 In this process you transferStandard the QRNA serial Fluotrac dilutions plate and from the add Standard diluted1 RNA RiboGreen. plate into the 2 3 Whole-Genome Gene Expression DASL HT Assay Guide Create Standard QRNA Plate with Diluted RiboGreen WG-DASL HT Assay Lab Protocols 50 rpr apeQN lt ihRbGenadRNA and RiboGreen with Plate QRNA Sample Prepare 2 and sample RNA contains 1 that plate QRNA Sample new a RiboGreen. create you process, this In 5 4 iue12 Figure d lo N apet l 6wlso h apeQN lt.Ol h first the Only plate. QRNA dilution. TE Sample RiboGreen/1X the of contain also 96 wells will all QRNA“. to columns “Sample two sample labelled RNA plate of Fluotrac the 2 µl of Add each 2 into and dilution 1 TE RiboGreen/1X columns of 195 µl well transfer pipette, multichannel a Using seal. aluminum to Proceed adhesive an with plate the cover Immediately tnadQN lt ihRiboGreen with Plate QRNA Standard rpr apeQN lt ihRbGenadRNA and RiboGreen with Plate QRNA Sample Prepare npg 50 page on at#1081 e.D Rev. 15018210 # Part . Quantitate RNA (Optional) 51 . . on page 51 Read QRNA Plate Sample QRNA Plate with RiboGreen NOTE For fewer than 96 RNAthe samples, number add of the wells diluted RiboGreen needed. reagent into Assays | Illumina | Illumina QRNA Immediately cover the plate with an adhesiveProceed to aluminum seal. Turn on the fluorometer. At the PC, openLoad the the SoftMax Illumina Pro QRNA.ppr program. filesystem. from the installation CD that cameSelect with your Figure 13 3 4 In this process, you useQRNA the and Gemini Sample XS QRNA or plates.the XPS The known Spectrofluorometer spectrofluorometer to concentrations creates read a in the standard theconcentration Standard Standard of curve RNA QRNA from plate, in which the you Sample use QRNA1 to plates. determine the 2 3 Whole-Genome Gene Expression DASL HT Assay Guide Read QRNA Plate WG-DASL HT Assay Lab Protocols 52 6 5 4 iue14 Figure Click 15 Figure to next arrow blue the well Click with corner. rack left loading fluorometer upper the the into in Plate A1 Fluotrac QRNA Standard the Place Read odteIlmn RAPooo nSfMxPro SoftMax in Protocol QRNA Illumina the Load eetteSadr N Screen RNA Standard the Select nteSfMxPoitraet ei edn h tnadQN Plate. QRNA Standard the reading begin to interface Pro SoftMax the in tnadRNA Standard . at#1081 e.D Rev. 15018210 # Part Quantitate RNA (Optional) 53 to view the standard curve graph. Standard Curve Read the Standard QRNA Plate When the software finishes reading the data,Click remove the the blue plate arrow from next the to drawer. Figure 16 7 8 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 54 11 10 9 iue17 Figure lc h learwnx to next left upper arrow blue the the in Click A1 well with fluorometer the in plate corner. QRNA Sample first the click Otherwise, Place plate. sample the with continue Curve acceptable, Standard is curve standard the If iwSadr Curve Standard View again. QRNA#1 n click and Read . at#1081 e.D Rev. 15018210 # Part Quantitate RNA (Optional) 55 and export on page 56 to save the output data file (*.pda). on page 60 File | Import/Export | Export File | Save to quantitate all Sample QRNA plates. 12 through Pre-Qualify cDNA Samples (Optional) Make Single-Use RNA (SUR) Plate 9 Read the Sample QRNA Plate Proceed to Proceed to Store the quantitated RNA at 2° to 8°C for up to one month. • • • When the software finishes reading the plate,Repeat remove steps the plate from the drawer. Once all plates have been read, click When you have saved thethe *.pda file file, as click a *.txt file. YouDo can one open of the the *.txt following: file in Microsoft Excel for data analysis. Figure 18 12 13 14 15 16 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 56 r-ulf DASmls(Optional) Samples cDNA Pre-Qualify Consumables minutes 10–60 time: Incubation ~15 minutes time: Hands-on Time Estimated cDNA, from only product amplified correctly a produce DNA. designed should genomic are and not primers intron These a an amplify NM_012423.2). span primers RPL13A to accession # RPL13A expressed (GenBank The highly fragment Green. the pair SYBR of by fragment 90 base detection a with of gene protein amplification ribosomal on based is process The your If qPCR. by analyzed 60 be to quality page proceed may RNA pre-qualification, require samples of not measure analysis, does relative cDNA a Assay degraded obtain HT partially WG-DASL To for to RNAs. degraded used prior entirely be for can not quality but poor of RNAs, Assay are replicates HT WG-DASL tissues run (FFPE) The paraffin-embedded to degraded. formalin-fixed, necessary commonly from is derived it RNAs if samples. determine RNA to you guides process This al 13 Table 6wl . lsitdmcolt lt per plate 1 microplate skirted 0.2 ml 96-well RNA Total Kit MCS4 Single-Use Synth cDNA from reagent RTE Kit Single-Use MCS4 Synth cDNA from reagent MCS4 3' GTTGGTGTTCATCCGCTTG 5' Reverse 3' GTACGCTGTGAAGGCATCAA 5' Forward Item Sequence Primer . rmr o PRuigSB re Detection Green SYBR using qPCR for Primers 0 gSemanufacturer’s See 24 samples 200 ng 24 samples per tube 1 24 samples per tube 1 uniyStorage Quantity aeSnl-s N SR Plate (SUR) RNA Single-Use Make instructions manufacturer’s See instructions -25°C to -15° -25°C to -15° at#1081 e.D Rev. 15018210 # Part User User User User By Supplied on Pre-Qualify cDNA Samples (Optional) 57 the 42°C before O. 2 NOTE Due to the small volume,the you tube. may wish to single-pipette directly out of NOTE Be sure to use RNase-freetips between materials and RNA techniques sample and dispenses. change pipette NOTE In the Pre-Qualify cDNA protocol,instead you of may a use 96-well three microplate. 8-well strip tubes incubation to prevent the wells from drying out. CAUTION It is important to centrifuge the SUR plate to 250 xg Add 5 µl MCS4 andnormalized RTE mixture RNA to each sample. well of the SURQuickly plate add that 5 µl will of contain normalizedcontaining a RNA 5 µl sample of to the each MCS4dispenses. well and of RTE the mixture. SUR Change plate tips betweenSeal RNA the sample SUR plate withsealed. a microplate heat seal. Ensure thatVortex all the wells sealed are plate completely at 2,300 rpmPulse for 20 seconds. centrifuge the samples to 250 xg for 1 minute. Normalize intact RNA samples tosamples 20–100 ng/µl to 40-200 ng/µl) (or partially with degraded DEPC-treated H RNA Preheat the heat sealer. Preheat a heat block to 42°CThaw and the MCS4 allow tube the to temperature to room stabilize. temperature. Add 32 µl RTE to 288 µl MCS4Pour and the entire mix contents well. of thedisposable MCS4 reservoir. and RTE tube mixture into a new, nonsterile, } } } 4 5 6 7 8 Steps 1 Preparation 2 3 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 58 11 10 9 13 12 us etiueteSRpaet 5 gfr1mnt ormv odnainfrom condensation remove to well. 1 minute each for of 250 xg walls to the plate reduce SUR 1 hour. to for the centrifuge lid 42°C Pulse the at close Incubate seal. and block plate heat the preheated on 1 hour). the condensation to on (up plate 10 minutes SUR the least Place at temperature room at plate SUR the Incubate P1Apiesadteraet itdaoewt eeto hehl e t0.2 at set threshold detection a with above listed reagents the for using and cycles primers catalog # 740000) 19 (Stratagene, RPL13A about RNA of Total Reference value (Ct) Human threshold Universal crossover a measures routinely Illumina cycler thermal the run and cycler For thermal PCR product. follows: the the as of into program uniformity plate the sealed the assess to place include example, available, and if instrument, curve, your for dissociation instructions a manufacturer’s the to µl. according Cycle 10 be should volume reaction total The System Detection Sequence contain: 7900HT should Prism reaction ABI the the Biosystems), a using (Applied as are product you cDNA if the example, of For dilution 1:10 a of using template. 1 µl instrument qPCR and your detection for Green appropriate SYBR reactions PCR duplicate the Assemble • • • 5 Mec owr n eev primers reserve and forward each nM 250 product cDNA - diluted Mix µl Master 1 PCR Green SYBR µl 5 ape a esoe fe h 2Cicbto o pt orhusat hours four to cDNA up -15° to -25°C. day, at for incubation same overnight 42°C the or the on 2° to 8°C step after qPCR stored the be can to samples proceeding not are you If NOTE 72°C 54°C 94°C 95°C eprtr Time Temperature minute 1 seconds 20 seconds 20 minutes 12 at#1081 e.D Rev. 15018210 # Part Pre-Qualify cDNA Samples (Optional) 59 Ct > 7 between the reference and any of the FFPE samples, then ∆ qPCR vs. WG-DASL: RPL13A 90nt ), and qPCR Ct values. 2 for the ABI Prism. We then(r compare the WG-DASL HT AssayWhen self-reproducibility the difference in Ct valuesamples between increases the beyond UHR seven, intact reproducibilitydeclines. RNA for As the sample a WG-DASL and HT result, FFPE Assay IlluminaqPCR recommends screen that with you RPL13A perform for aas all pre-qualification a FFPE samples reference. If alongrunning with technical an replicates intact for RNA those sampleDASL samples HT is Assay results recommended to will ensure be the reliable. Figure WG- 19 If you use other qPCRgeneral, systems, if you the may difference expect in aparaffin-derived cycle RNAs variation number exceeds at from 7 these detection between cycles, values,samples cryo-preserved then and but is running in recommended. technical replicates for those Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 60 aeSnl-s N SR Plate (SUR) RNA Single-Use Make Consumables incubation 1-hour one incubation, 10-minute One time: Incubation ~15 minutes time: Hands-on Time Estimated used be to sample individual each from RNA once sufficient transcribes reverse process This iue20 Figure microplate skirted 0.2 ml 96-well samples RNA reagent RTE reagent MCS4 Item Assay. HT WG-DASL the in nteMk U lt rtcl o a s he -elsrptubes strip 8-well three use microplate. 96-well may a you of protocol, instead Plate SUR Make the In NOTE once. reagent at enough samples provides 96 WG- kit 96 sample prepare the a to Kit, using Reagent are Profiling you Assay If HT DASL Kit. WG- Reagent Profiling 24 sample Assay a HT using DASL 24 samples preparing describe procedures These NOTE aeSUR Make 24 samples per plate 1 24 24 samples per tube 1 24 samples per tube 1 Quantity instructions manufacturer’s See -80°C -25°C to -15° -25°C to -15° Storage at#1081 e.D Rev. 15018210 # Part User User Illumina Illumina By Supplied Make Single-Use RNA (SUR) Plate 61 the 42°C before O. 2 NOTE Due to the small volume,the you tube. may wish to single-pipette directly out of NOTE Be sure to use RNase-freeSUR process. materials and techniques throughout the Make incubation to prevent the wells from drying out. CAUTION It is important to centrifuge the SUR plate to 250 xg Incubate the SUR plate at room temperature at least 10 minutes (up to 1 hour). Add 5 µl MCS4 andplate. RTE mixture to each well of columnsQuickly 1, add 2, 5 µl and 3 of normalized ofthe the RNA SUR SUR sample plate. Change to tips each between well RNA ofSeal sample columns the dispenses. 1, SUR 2, plate and withsealed. a 3 microplate of heat seal. Ensure thatVortex all the wells sealed are plate completely at 2,300 rpmPulse for 20 seconds. centrifuge to 250 xg for 1 minute. Preheat the heat sealer. Preheat a heat block to 42°CThaw and the MCS4 allow tube the to temperatureApply to room a stabilize. temperature. SUR barcode label to a new 96-well microplate. Normalize intact RNA samples tosamples 20–100 ng/µl to 40-200 ng/µl) (or partially with degraded DEPC-treated H RNA Add 32 µl RTE to the MCS4Pour tube (288 µl) the entire and contents mix of thedisposable well. MCS4 reservoir. and RTE tube mixture into a new, nonsterile, } } } } 9 4 5 6 7 8 Preparation Steps 1 2 3 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 62 12 11 10 ooeo h following: the of from one Do condensation remove to well. 1 minute each for of 250 xg walls to the plate reduce SUR 1 hour. to for the centrifuge lid 42°C Pulse the at close Incubate seal. and block plate heat the preheated on the condensation on plate SUR the Place • • orhusa °t ° ru o2 or t-15° to -25°C. at 24 hours to up or 2° to 8°C at hours four to immediately proceed to Plate plan not do you If to proceed and Plate 70°C (ASE) to Extension block heat a set Immediately npg 63 page on hnsoetesae U lt fe h 2Cicbto pto up incubation 42°C the after plate SUR sealed the store then , npg 63 page on tr hwn h A n B reagents. OB1 and DAP the thawing Start . aeAsySeii xeso (ASE) Extension Assay-Specific Make aeAssay-Specific Make at#1081 e.D Rev. 15018210 # Part Make Assay-Specific Extension (ASE) Plate 63 Supplied By Illumina Illumina User Storage -15° to -25°C 2° to 8°C or -15° to -25°C See manufacturer’s instructions Quantity (per SUR plate) 1 tube 1 tube 1 plate Make ASE NOTE These procedures describe preparing 24 samplesDASL using HT a Assay 24 sample Profiling Reagent WG- Kit.DASL HT If Assay you Profiling are Reagent using Kit,to a the prepare 96 sample kit WG- 96 provides samples enough at reagent once. Item OB1 reagent DAP reagent 96-well 0.2 ml skirted microplate Figure 21 Estimated Time Hands-on time: ~30 minutes Incubation time: 14–20 hours Consumables This process combines thehybridization biotinylated cDNAs reagents, with and Assay-specific paramagnetic oligos particles(ASE) (ASOs), plate. in The an plate Assay istarget Specific then of Extension placed interest are in allowed asimultaneously to heat anneal captured block by to and paramagnetic the the particles. biotinylatedthe ASOs cDNA The extension for samples. resulting and each ASE The ligation sequence plate cDNA of is is the ready hybridized for oligos on the bound cDNAs. Whole-Genome Gene Expression DASL HT Assay Guide Make Assay-Specific Extension (ASE) Plate WG-DASL HT Assay Lab Protocols 64 Preparation 6 5 4 3 2 1 Steps } } } } } } } upne nslto.Pu h niecnet fteO1tb noasterile a into tube OB1 the of evenly contents are entire reservoir. particles the Pour paramagnetic solution. all in that the suspended verify resuspend to completely tube to the Vortex temperature. Invert room solution. reservoir. to sterile tube a tube. OB1 into the the tube of Thaw bottom the of the mix contents at entire to contents the contents the Pour the collect vortex to centrifuge and temperature pulse room then completely, to tube temperature reagent room DAP to the 1 minute. it for Thaw thaw 250 xg overnight, to -15° to -25°C pulse-centrifuge at then stabilize. stored to and temperature was the plate allow SUR the and If 70°C to block heat the Preheat Sample_Well. sealer. heat each for the DAP Preheat the enter see Sheet, information, Sample more the For of column Pool_ID the In us etiueteAEpaet 5 gfr1 minute. for 250 xg to plate ASE are the wells centrifuge all Pulse that Ensure sealer. heat microplate sealed. a completely with plate ASE the Heat-seal the to plate SUR plate. the ASE of the well of occupied well each corresponding from cDNA biotinylated splashing 10 µl avoid Transfer to plate. care ASE Take the plate. of SUR wells. the 3 the from from and seal 2, plate. heat 1, ASE the the columns remove of of Carefully well 3 each and to 2, OB1 1, 30 µl columns Add of well each to DAP 10 µl Add microplate. 96-well new a to label barcode ASE an Apply ontcnrfg h B tube. OB1 the centrifuge not Do CAUTION rnfrteetr otnso ahwl rmteSRpaet h ASE the to plate SUR the from well each plate. of contents entire the Transfer NOTE apeSheet Sample npg 31 page on . at#1081 e.D Rev. 15018210 # Part Make Assay-Specific Extension (ASE) Plate 65 . on page 66 Add Master Mix for Extension & Ligation (MEL) Vortex the ASE plate atresuspended. 1,600 rpm for 1 minute or until allPlace beads the are sealed completely ASE plate on theImmediately preheated change 70°C the heat set block temperature and ofplate the close in heat the the block lid. heat to block 30°C. for Leave 14–20 hoursProceed the to ASE while it cools to 30°C. 7 8 9 10 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 66 d atrMxfrEtnin&Lgto (MEL) Ligation & Extension for Mix Master Add nuaintm:15 minutes time: Incubation ~45 minutes time: Hands-on Time Estimated and extension The sample. 45°C. cDNA of at each to (consisting occurs (MEL) added reaction ligation mix is enzymes) master are ligation ligation oligos and and excess extension extension and an mis-hybridized Next, cDNA, away. the to washed hybridized are oligos the After iue22 Figure h elti uldfrtt n ieadte oteother. the to then that and so side bars, one magnetic to the first pulled also over is can pellet forth you and the necessary, back rapidly If the plate pellet. Follow the the resuspend. shuttle up to difficult break be to instructions may pellet vortexing bead the process, this In CAUTION opeae9 ape tonce. reagent at enough samples provides 96 WG- kit 96 sample prepare the a to Kit, using Reagent are Profiling you Assay If HT DASL Kit. WG- Reagent Profiling 24 sample Assay a HT using DASL 24 samples preparing describe procedures These NOTE d MEL Add at#1081 e.D Rev. 15018210 # Part Add Master Mix for Extension & Ligation (MEL) 67 Supplied By Illumina Illumina a Storage 2° to 8°C 2° to 8°C -15° to -25°C Illumina Bottle Bottle 1 tube per ASE plate NOTE To avoid tip contamination andthe sample loss pipette during tips so this procedure, thatopposite slant they the draw beads. liquid from the side of the well Item Quantity -15°to -25°C for long-term storage AM1 reagent UB1 reagent MEL reagent Immediately place the ASE plateuntil on the the beads raised-bar are magnetic completely plate captured. for 2 minutes or a Thaw the MEL tube tosterile room reservoir temperature. right Pour before the using entire it. contentsRemove of the the AM1 tube into bottle from a 10 minutes. the refrigerator and Pour 11 ml leave it AM1additional at into plate. room a temperature second for sterile reservoir.Remove Add the 10 ml UB1 for bottle from each thereservoir. refrigerator. Pour 11 ml UB1 intoRemove a the third IP1 sterile and SCM tubes from the freezer and let them thaw. Remove the ASE plate from the heatCentrifuge block the and ASE plate reset the to heat 250 xg. block to 45°C. } } } } 3 1 2 Preparation Consumables Whole-Genome Gene Expression DASL HT Assay Guide AM1 Washes WG-DASL HT Assay Lab Protocols 68 5 4 10 9 8 7 6 cuidwlsaddsadi.Laetebasi h wells. the the in from beads (~50 µl) the liquid Leave the it. all discard remove and tips, wells new occupied with pipette sample 8-channel splash an to Using not care Take plate. wells. ASE the the of from out seal heat the remove Carefully eoetesa rmteAEpae aigcr oaodslsigfo h wells. the from splashing avoid to care taking plate, ASE the from seal or the Remove 2 minutes approximately captured. for completely plate are resuspended. magnetic beads are raised-bar the beads the until all on until plate or ASE the 20 seconds Place for 1,600 rpm at plate ASE film. the Vortex adhesive clear with plate ASE the Seal plate. ASE the of well occupied with each pipette 23 to Figure 8-channel AM1 an use 50 µl plate, add magnetic to raised-bar tips the new on liquid plate the ASE removed the With have you until tips. pipette again the 3 columns. tips change pipette all and change from solution to beads, the re-collect need return to not tips, magnet do pipette the You in allow visible wells, ensure are to same beads the column If to each removed. from been liquid have removing beads after no tips pipette the inspect Visually gis h o deo h well. tips the the of place tips, edge the top contaminating the or against pellet the disturbing avoid To NOTE vi i Contamination Tip Avoid at#1081 e.D Rev. 15018210 # Part Add Master Mix for Extension & Ligation (MEL) 69 . on page 70 Make PCR Plate once. once. 11 4 through through 6 1 CAUTION Do not allow the ASE plate to incubate at 45°C for any longer than 15 minutes. Using the same 8-channel pipetteeach with occupied the well. same Leave tips, theYou remove beads all do in not AM1 the need wells. reagent to from from change all pipette tips 3 columns. again until you haveRepeat steps removed the liquid Remove the ASE plate from the raised-barUsing magnetic an plate. 8-channel pipette withthe new ASE tips, plate. add 50 µl UB1Place to each the ASE occupied plate well onto of or the until raised-bar the magnetic beads plate are for completely approximately captured. 2 minutes Using the same 8-channel pipetteeach with occupied the well. same Leave tips, theYou remove beads all do in not UB1 the need reagent wells. to from from change all pipette tips 3 columns. again until you haveRepeat steps removed the liquid Using an 8-channel pipette withthe new ASE tips, plate. add 37 µl MELSeal to each the plate occupied with well clear of adhesive film. Vortex the plate at 1,600 rpm for 1 minuteIncubate the to ASE resuspend plate the on beads. During the the preheated incubation, 45°C perform heat block for exactly 15 minutes. 11 12 1 2 3 4 5 1 2 3 4 Whole-Genome Gene Expression DASL HT Assay Guide UB1 Washes Add MEL WG-DASL HT Assay Lab Protocols 70 aePRPlate PCR Make ad-ntm:~15 minutes time: Hands-on Time Estimated the for plate 24-sample a creates It PCR. for process. Uracil mix PCR optional master Inoc the SCM the and to Polymerase DNA Glycosylase Illumina-recommended DNA the adds process This iue24 Figure opeae9 ape tonce. reagent at enough samples provides 96 WG- kit 96 sample prepare the a to Kit, using Reagent are Profiling you Assay If HT DASL Kit. WG- Reagent Profiling 24 sample Assay a HT using DASL 24 samples preparing describe procedures These NOTE ae nclt,adCcePCR Cycle and Inoculate, Make, at#1081 e.D Rev. 15018210 # Part Make PCR Plate 71 Supplied By User User Illumina User -15° to -25°C -15° to -25°C -15° to -25°C instructions Quantity Storage Tube Tube 1 tube per PCR plate 1 per ASE plate See manufacturer’s . on page 72 Item Illumina-recommended DNA Polymerase Uracil DNA Glycosylase (Optional) SCM reagent 96-well 0.2 ml skirted microplate Apply a PCR barcode labelInvert to the a thawed new SCM 96-well tube 0.2 ml 10 times skirted to microplate. mix. Add 800 µl SCM andml 16 µl tube. Illumina-recommended DNA Polymerase to aAdd clean 12.5 µl 1.5 Uracil DNA glycosylase to theInvert SCM/Polymerase the mixture. tube several timessterile to reservoir. mix the contents and pipette theUsing contents an into 8-channel a pipette, add1, 30 µl 2, and of the 3 SCM of mixture the to PCRSeal each plate. the well PCR of plate columns with clear adhesiveAs film. soon as the 15 minuteInoculate PCR ASE Plate plate incubation is complete, proceed immediately to } } Steps 1 2 3 4 5 6 Preparation Consumables Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 72 nclt C Plate PCR Inoculate Preparation Consumables ~30 minutes time: Hands-on Time Estimated eluted. be to signal fluorescent PCR the the containing a captures strand primer with the biotinylated labeled The allows is biotinylated. and One a is product primers. other in the universal process two and ligation dye uses and fluorescent reaction extension PCR the This during reaction. formed template PCR the uses process This } } a reservoir. sterile second a into tube IP1 the of contents entire reservoir. the sterile Pour a into UB1 6 ml Pour 1°t 2° o ogtr storage long-term for -25°C to -15° reagent IP1 reagent UB1 tmQuantity Item h elti uldfrtt n ieadte oteother. the to then that and so side bars, one magnetic to the first pulled also over is can pellet forth you and the necessary, back rapidly If the plate pellet. Follow the the resuspend. shuttle up to difficult break be to instructions may pellet vortexing bead the process, this In CAUTION opeae9 ape tonce. reagent at enough samples provides 96 WG- kit 96 sample prepare the a to Kit, using Reagent are Profiling you Assay If HT DASL Kit. WG- Reagent Profiling 24 sample Assay a HT using DASL 24 samples preparing describe procedures These NOTE plate PCR per tube 1 Bottle 1°t 2° Illumina -25°C to -15° 8°C to 2° Storage a Illumina By Supplied at#1081 e.D Rev. 15018210 # Part Inoculate PCR Plate 73 NOTE To avoid tip contamination andthe sample loss pipette during tips so this procedure, thatopposite slant they the draw beads. liquid from If you thethe side suspect contents of that of the the the tips well are well, discard contaminated the with tips and use new ones. NOTE The amount of supernatant insetting this the step pipette is to less than thatlater 50 µl. volume washes, However, ensures which require that the it is full 50 µl. set correctly for the Visually inspect the pipette tipsno after beads removing have liquid been from removed. eachto If column the beads same to are ensure wells, visible allow inYou the pipette do magnet tips, not to return need re-collect the beads, to solution from change and all pipette change tips 3 columns. the again pipette tips. until you have removed the liquid Leaving the plate on the50 µl magnet and UB1 to using each an occupied 8-channel well pipetteLeave of with the the new ASE ASE tips, plate. plate add onbeads the are raised-bar completely magnetic captured. plate for 2 minutesRemove or until and the discard the supernatantplate. (~50 µl) Leave the from beads all in occupied the wells wells. of the ASE Remove the ASE plate from the heatReset block. the heat block to 95°C. Place the ASE plate onbeads the are raised-bar completely magnetic captured. plate for 2 minutesRemove or the until clear the adhesive film from theUsing plate. an 8-channel pipette, removeoccupied and wells discard of the the supernatant ASE plate. (~50 µl) Leave from the all beads in the wells. 1 2 3 1 2 3 4 5 Whole-Genome Gene Expression DASL HT Assay Guide UB1 Wash Remove Supernatant WG-DASL HT Assay Lab Protocols 74 d uentn oPRPlate PCR to Supernatant Add IP1 Add 6 5 4 3 2 1 5 4 3 2 1 o ontne ocag iet isutlyuhv eoe h iudfo all from liquid the removed have you until tips pipette change 3 columns. to need not do You meitl rnfrtePRpaet h hra ylr icr h S plate. ASE the Discard cycler. to Proceed thermal the to plate PCR the transfer 1 minute. Immediately for 250 xg to plate the centrifuge thermal Pulse your for film plate-sealing PCR appropriate the cycler. with plate PCR the Seal times. 3–4 down dispenses. Pipette and plate. column between up PCR tips the wells of Change plate well PCR corresponding the each the of from to contents supernatant plate the ASE 30 µl the transfer of tips, well new occupied with pipette 8-channel an Using plate. PCR the from seal the Remove the until or 2 minutes for plate captured. magnetic completely raised-bar are the beads on 1 minute. plate for ASE block the heat Place 95°C preheated resuspended. the are on beads plate all the until Place or 1 minute for 1,800 rpm at Vortex film. adhesive clear of well with occupied plate the each to Seal IP1 35 µl add plate. tips, ASE new the with pipette 8-channel an Using aeseilcr o odsubo rnfrtebaswe siaigthe aspirating when beads the transfer or product. disturb eluted to not care special Take CAUTION the on cross-contamination. evaporation cause the and drip that not so carefully does seal very seal adhesive the Remove CAUTION hra yl C Plate PCR Cycle Thermal npg 76 page on . at#1081 e.D Rev. 15018210 # Part Inoculate PCR Plate 75 This concludes the Pre-PCR processesAssay. for If the you Whole-Genome remove Gene materials Expressionnot such DASL return with as them experienced user in cards to the from Pre-PCR the lab Pre-PCR lab, at do any time. Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 76 hra yl C Plate PCR Cycle Thermal 2 14 Table 1 Steps 45 minutes ~2 hours time: Cycle Time Estimated the amplify and label process. fluorescently pre-PCR to the plate in PCR generated the templates cycles thermal process This ooeo h following: the of one Do program cycler thermal the run table. and cycler this thermal in the shown into plate sealed the Place • • eladsoetePRpaea -15° to -25°C. at plate PCR the store and Seal to immediately Proceed hra ylrProgram Cycler Thermal 2C1 minutes 10 minutes 2 4°C seconds 35 72°C seconds 35 72°C minutes 3 minutes 56°C 10 95°C 95°C 37°C eprtr Time Temperature minutes 5 idPRProducts PCR Bind npg 77 page on . at#1081 e.D Rev. 15018210 # Part Bind PCR Products 77 Supplied By Illumina User Storage 2° to 8°C instructions Quantity plate 1 per PCR plate See manufacturer’s Bind PCR Products NOTE These procedures describe preparing 24 samplesDASL using HT a Assay 24 sample Profiling Reagent WG- Kit.DASL HT If Assay you Profiling are Reagent using Kit,to a the prepare 96 sample kit WG- 96 provides samples enough at reagent once. Item MPBreagentFilter plate with 1tubeperPCRlid Figure 25 Estimated Time Hands-on time: ~20 minutes Incubation time: 1 hour Consumables In this step, the double-strandedbiotinylated PCR strand products to are paramagnetic immobilized particles. byand The binding incubated solution the at is room transferred temperatureparamagnetic to so a particles. that filter the plate PCR product may bind to the Whole-Genome Gene Expression DASL HT Assay Guide Bind PCR Products WG-DASL HT Assay Lab Protocols 78 Preparation 3 2 1 Steps } } pl h itrpaelblt h o ftefle lt ett column 12. to next plate filter label. the plate 26 of filter Figure top the the on to provided label space plate the filter reservoir. the in sterile Apply number barcode a plate into resuspended. tube PCR completely the MPB are Write the beads of the contents until entire or the times Pour several tube MPB the Vortex ltsnx oec te ihteA el nteuprlf corner. filter left and upper PCR the the in Place wells 85 µl. A1 to the it with set other each and pipette to next 8-channel plates an on tips new Place all to transferred been has liquid until tips pipette plate. change PCR 3 columns. to the resuspended of necessary well not 20 µl occupied transfer is It each and pipette into reservoir multichannel the from 5–50 µl MPB a onto tips 1 minute. new for Place 250 xg to plate PCR the centrifuge Pulse ftewl,dsadtetp n s e ones. contents new the use with and tips contaminated opposite well the are discard the tips well, the of the side that of suspect the you from liquid If draw beads. they slantthe that procedure, this so tips during pipette loss sample the and contamination tip avoid To NOTE pl ae oFle Plate Filter to Label Apply at#1081 e.D Rev. 15018210 # Part Bind PCR Products 79 . on page 80 Make Intermediate (INT) Plate Pipette the solution in thewith PCR the plate PCR up product. and Transfer downPCR the several plate mixed into times solution the from to corresponding each mix wellChange occupied the of pipette well beads tips the of filter between the column plate. dispenses. Discard the empty PCR plate. Cover the filter plate with the filter plateStore at lid. room temperature, protected from light, for 1 hour.Proceed to 4 5 6 7 8 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 80 aeItreit IT Plate (INT) Intermediate Make 1 Steps Preparation Consumables ~20 minutes time: Hands-on Time Estimated plate. (INT) is intermediate plate an filter into the eluted from then product and PCR fluor-labeled washed single-stranded the step, this In } } } } lc h itrpaeaatro nepy naee 6wl -otmpae(waste plate V-bottom 96-well unlabeled empty, an on plate). adapter plate filter the Place reservoir. sterile third a into tube MH1 an reservoir. of sterile contents second the reservoir. Pour a sterile into a into 0.1N NaOH microplate. UB2 5 ml skirted Pour 10 ml 0.2 ml transfer pipette, 96-well serological new a a Using to label barcode INT a Apply 6wl -otmpae1prfle lt e manufacturer’s See plate filter per adapter 1 plate Filter microplate skirted 0.2 ml 96-well plate V-bottom 96-well reagent MH1 reagent UB2 NaOH 0.1N Item opeae9 ape tonce. reagent at enough samples provides 96 WG- kit 96 sample prepare the a to Kit, using Reagent are Profiling you Assay If HT DASL Kit. WG- Reagent Profiling 24 sample Assay a HT using DASL 24 samples preparing describe procedures These NOTE e itrplate filter per 1 plate filter per 1 plate INT per tube 1 Bottle Bottle uniyStorage Quantity omtmeaueIllumina Illumina instructions temperature Room temperature Room 8°C to 2° at#1081 e.D Rev. 15018210 # Part User User User User By Supplied Make Intermediate (INT) Plate 81 Assemble Filter Plate CAUTION Be sure to replace thethe waste waste plate plate with will the result in INT loss plate. Failure of to samples. replace CAUTION To avoid disturbing the pelletagainst or the contaminating top the edge tips, place of the the tips well. Place adapter on waste plate Place filter plate on adapter Completed Assembly A B C Centrifuge to 1000 xg for 5 minutes at 25°C. Remove the filter plate lid. Using an 8-channel pipette with2, new and tips, 3 add of 50 µl the filter UB2 plate. to Dispense each slowly well to of avoid columns disturbing 1, the beads. Replace the filter plate lid. Centrifuge to 1000 xg for 5 minutes at 25°C. Using an 8-channel pipette with1, new 2, tips, and add 3 30 µl of the MH1 INTReplace to plate. the each waste well plate of with columns the the filter INT plate plate. matches Orient well the A1 INT plate of so the INT that plate. well A1 of Place the filter plate containingadapter. the bound PCR products onto the filterFigure plate 27 3 4 5 6 7 8 9 2 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 82 12 11 10 17 16 15 14 13 elc h itrpaelid. plate occupied filter each the to Replace 0.1N NaOH 30 µl plate. filter add the tips, of new well with pipette 8-channel an Using plate. waste the Discard elteITpaewt 6wl a a.Soetepaei h akutlrayto ready until following: dark the the of in one plate Do the Store mat. samples. of cap precipitation 96-well with a proceed with without plate side INT to the side Seal from it moving by plate INT protocols. the splashing. other of in contents use the later mix for Gently adapter the Save plate. plate. filter INT the the beads Discard of no end, wells the the At in 25°C. visible at be 5 minutes should for 1000 xg to immediately Centrifuge • • fyud o lnt s h N lt meitl ntepooo,soei at it store protocol, the in immediately plate 24 hours. INT to the up use for to -15° to -25°C plan not do you If to Proceed ufcet h N sdntrdams instantly.) almost is denatured 5 minutes is quickly. than DNA less proceed The NaOH, sufficient. unnecessary; 0.1N is NaOH to with dyes incubation the Prolonged of sensitivity the to Due CAUTION rcptt n ahITPlate INT Wash and Precipitate npg 83 page on . at#1081 e.D Rev. 15018210 # Part Precipitate and Wash INT Plate 83 Supplied By User Storage 2° to 8°CRoom temperature Illumina 3.2 ml per INT plate Hybridize to BeadChip NOTE These procedures describe preparing 24 samplesDASL using HT a Assay 24 sample Profiling Reagent WG- Kit.DASL HT If Assay you Profiling are Reagent using Kit,to a the prepare 96 sample kit WG- 96 provides samples enough at reagent once. Item Quantity PS1 reagent 2-propanol Bottle Figure 28 Estimated Time Hands-on: ~1 hour Consumables In this step the single-strandedresuspended. product The from product the from INT this plate plate is is precipitated, hybridized washed to and the BeadChip. Whole-Genome Gene Expression DASL HT Assay Guide Precipitate and Wash INT Plate WG-DASL HT Assay Lab Protocols 84 2 1 Steps Preparation } } } } } } } } } d 0µ S egn oec elo h N plate. INT the wells. of the well from each splashing to reagent avoid PS1 to care 30 µl taking Add plate, INT the from seal the Remove reservoir. sterile fourth centrifugation. a pulse into by mix followed water nuclease-free vortexing, MH1/water/HYB by the 300 µl well Pour MH1, Mix HYB. 300 µl combine 1.2 ml tube centrifuge and 15 ml reservoir. sterile sterile a third In a into reservoir. 70% EtOH sterile 20 ml second Pour a reservoir. into sterile 2-propanol a into stabilize. 10 ml to Pour 3.2 ml temperature transfer the then allow bottle and PS1 Vortex 65°C using. to before thoroughly for block 58°C may heat mix at that a and temperature incubate Preheat salts room undissolved, any to remain Cool dissolve to salts 10 minutes. any another 10 minutes If for storage. oven in 58°C precipitated the have in tube HYB to the it Place for 30 minutes Allow 58°C. to equilibrate. Oven Hybridization Illumina the Preheat digital system. Plus your Full-Scale with the supplied with thermometer Oven Hybridization Illumina the Calibrate H 0 lprINT per µl 300 reagent HYB MH1 Bottle EtOH 70% tmQuantity Item o orrgo.Frmr nomto,seteMD o hskt hc is which kit, this for MSDS the see information, http://www.illumina.com/msds. at more available standards For safety region. your governmental any for the and with containers accordance of in Dispose contents contact. unused eye and inhalation, probable contact, through a skin occur is ingestion, can that injury amide aliphatic Personal an toxin. of reproductive use the involves protocol This WARNING plate INT per ml 1.2 plate 1°t 2° Illumina -25°C to -15° temperature Room temperature Room Storage Illumina User By Supplied at#1081 e.D Rev. 15018210 # Part Precipitate and Wash INT Plate 85 CAUTION Do not tilt the plate,Tap as the this plate can firmly cause enough cross-contaminationdoes between to not decant wells. work all the as supernatant; well. tapping lightly CAUTION Do not tilt the plate,Tap as the this plate can firmly cause enough cross-contaminationdoes between to not decant wells. work all the as supernatant; well. tapping lightly CAUTION It is important to mixprecipitation the of contents the thoroughly DNA pellet. to ensure efficient Tap the inverted plate onto the pad to blot excess supernatant. Tap the inverted plate onto the padAdd to 150 µl blot excess 70% EtOH supernatant. to each wellUsing of a the INT multichannel plate. pipette, thoroughlypipetting wash up the and blue down pellet several in times. 70% EtOHSeal by the INT plate with clear adhesiveCentrifuge the film. plate to 3000 xg at 2° to 8°CRemove for the 10 minutes. INT plate from the centrifuge. Remove the INT plate sealand and smacking decant it the down supernatant onto by an inverting absorbent the pad. INT plate Seal the INT plate with clear adhesiveCentrifuge the film. plate to 3000 xg at 2° to 8°CRemove for the 20 minutes. INT plate from the centrifuge. Remove the INT plate sealand and smacking decant it the down supernatant onto by an inverting absorbent the pad. INT plate Using a multichannel pipette, thoroughlyup mix and the down contents several by pipetting times the until solution Add the 90 µl solution is 2-propanol uniformly to blue. each wellUsing of the a INT multichannel plate. pipette, thoroughlyup mix and the down contents several by pipetting times the until solution the solution is uniformly blue. 17 10 11 12 13 14 15 16 6 7 8 9 3 4 5 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 86 26 25 24 23 22 21 20 19 18 elteITpaewt 6wl a a.Soetepaei h akutlrayto ready until following: dark the the of in one plate Do the Store BeadChip. mat. a cap onto 96-well sample a dispense with plate INT the Seal times. the pipetting several by down pellets and the up dissolve solution thoroughly pipet, multichannel a Using seal. plate INT the Remove 250 xg. to plate the centrifuge film. Pulse adhesive plate. clear INT with the of plate INT well the each Seal to mix MH1/water/HYB the of has EtOH 15 µl Add residual the until or 5 minutes for lid. 65°C evaporated. the at close plate and INT block the heat Incubate preheated the in plate INT the Place • • fyud o lnt s h N lt meitl ntepooo,soei at it store protocol, the in immediately plate 24 hours. INT to the up use for to -15° to -25°C plan not do you If to Proceed yrdz BeadChip Hybridize npg 87 page on . at#1081 e.D Rev. 15018210 # Part Hybridize BeadChip 87 . on page 31 Supplied By Illumina Sample Sheet -15° to -25°CRoom temperature Illumina Quantity Storage 4 BeadChips 2 per 24 samples 2° to 8°C Illumina NOTE These procedures describe preparing 24 samplesDASL using HT a Assay 24 sample Profiling Reagent WG- Kit.DASL HT If Assay you Profiling are Reagent using Kit,to a the prepare 96 sample kit WG- 96 provides samples enough at reagent once. Item HCB reagentHyb Chamber Tube 1 per BeadChips (12x1) Calibrate the Illumina Hybridization Oventhermometer with supplied the with Full-Scale your Plus system. digital Preheat the Illumina Hybridization Ovenequilibrate. to 58°C. Allow 30 minutes forPlace it the to HCB tube inhave the precipitated 58°C in oven storage. for If 10 minutesanother any 10 minutes. to salts dissolve Cool remain to any undissolved, room salts incubate temperature and that at may mix 58°C for thoroughly before using. In the Sentrix_ID column ofBeadChip the section. Sample For Sheet, more enter information, the see BeadChip ID for each } } } } Preparation Estimated Time Hands-on time: ~30 minutes Incubation time: 14–20 hours Consumables and Equipment In this process the BeadChipsChamber has are hybridized been using assembled, the thehybridized Hyb samples overnight Chamber. in are After the the ready Hyb Illumina for hybridization. Hybridization The Oven BeadChip at is 58°C. Whole-Genome Gene Expression DASL HT Assay Guide Hybridize BeadChip WG-DASL HT Assay Lab Protocols 88 sebeHbiiainChambers Hybridization Assemble 1 } fteITpaehsbe rzn hwi opeeya omtmeauei light- 1 minute. a for in 250 xg temperature to room it at centrifuge completely pulse it then thaw and frozen, drawer, been protected has plate INT the If iue29 Figure top: bench the on items following the Place • • • edhpHbCabrisrs( e y Chamber) Hyb per (4 inserts Chamber Chamber) Hyb Hyb BeadChip per (1 gasket Chamber BeadChips) Hyb BeadChip 4 per (1 Chamber Hyb BeadChip C B A y hme Inserts Chamber Hyb Gasket Chamber Hyb Chamber Hyb h vn fsml vprto rohrlbpoesn anomalies. processing lab other in evaluated or and for evaporation investigated used sample was be of can that event Chambers Chamber the Hyb Hyb that the so Record BeadChip, base. the seal each the airtight that and an important lid is ensure the It to fittings. between strength loose clamping regularly or adequate hinges wear provide Check hinges abnormal secure. of remains signs fit lid-base any the for Chamber that Hyb ensure Check to lid. Hyb regularly original each pairs its lids keeps with that paired Chamber convention base Hyb labeling the Chamber a Adopt keep together. Chambers bases Hyb and from results optimal ensure To NOTE edhpHbCabrComponents Chamber Hyb BeadChip at#1081 e.D Rev. 15018210 # Part Hybridize BeadChip 89 Placing Gasket into Hyb Chamber Hyb Chamber and Gasket Reservoirs Barcode Ridges Narrower Edges Wider Edges A B C D Make sure the Hyb Chamber gaskets are properly seated. Lay the gasket into the Hyb Chamber, and then press it down all around. Match the wider edge ofthe the Hyb Hyb Chamber. Chamber gasket to the barcode-ridge side of c b Figure 31 Place the BeadChip Hyb Chambershown. gaskets into the BeadChip Hyba Chambers as Figure 30 2 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 90 4 3 iue32 Figure ls n okteBaCi y hme lid. Chamber Hyb a BeadChip the lock and Close BeadChips. contain 33 Chamber. will Figure Hyb that the sections in of reservoirs reservoirs buffer the humidifying fill eight Only the into HCB 200 µl Add etteldscrl ntebto plate. bottom the on securely lid the Seat y hme ihGse nPlace in Gasket with Chamber Hyb ipnigHBit y hme Reservoir Chamber Hyb into HCB Dispensing at#1081 e.D Rev. 15018210 # Part Hybridize BeadChip 91 Place BeadChips into Hyb Chamber Inserts Sealing the Hyb Chamber CAUTION Do not unpackage BeadChips unless you are ready to begin hybridization. Snap two clamps shut, kitty-cornerSnap across the from other each two other. clamps. Leave the closed Hyb ChambersBeadChips on are the loaded bench with at DNA room sample. temperature until the Remove all the BeadChips from their packages. Place each BeadChip in amatches Hyb the Chamber barcode Insert, symbol orienting on the the barcode Hyb endFigure Chamber so 35 Insert. that it b c Figure 34 1 2 5 Whole-Genome Gene Expression DASL HT Assay Guide Prepare BeadChip for Hybridization WG-DASL HT Assay Lab Protocols 92 odSample Load 4 3 2 1 iue37 Figure Hyb each into BeadChips sample-laden Chamber. containing Inserts Chamber 4 Hyb normal. Load is This port. Chamber. inlet Hyb the the in Open remain stripe. still the may of sample sections covered. residual the not Some of are all that covers sections sample any Ensure Record sections. all inspect Visually 36 Figure each of center the onto sample 15 µl port. add inlet pipette, precision single-channel a Using sso salary aercie sample. received have arrays all step as next To the soon surface. as to array immediately the proceed to contamination/evaporation, tips avoid pipette placing directly by samples Load NOTE edhp nBaCi y Chamber Hyb BeadChip in BeadChips BeadChip onto Sample Dispense at#1081 e.D Rev. 15018210 # Part Hybridize BeadChip 93 Secure Lid Seat the lid securely onSnap the bottom two plate. clamps shut, diagonallySnap across the from other each two clamps. other. Place the Hyb Chamber into the 58°CStart Illumina the Hybridization rocker Oven. by turning onswitch the (optional). switch just above the power Incubate for 16 hours at 58°C. In preparation for the nextthe day’s 10X washes, stock prepare by 1X High-Temp adding Washwater. 50 ml buffer from 10x High-Temp Wash buffer to 450 mlPlace the nuclease-free Hybex Waterbath insert into theAdd Hybex Heating 500 ml Base. prepared 1X High-Temp Wash buffer to the Hybex Waterbath insert. Position the barcode end overthe the inserts ridges indicated are securely on seated. the Hyb Chamber and ensure Close and lock the BeadChipa Hyb Chamber lid. b c Figure 38 2 3 4 5 6 7 5 1 Whole-Genome Gene Expression DASL HT Assay Guide Hybridize BeadChips WG-DASL HT Assay Lab Protocols 94 10 9 8 iue39 Figure ls h ye etn aeldadlaeteHg epWs ufrt warm to buffer to Wash Proceed Temp High the leave and lid overnight. Base Heating Hybex the Close 55°C. to temperature Base Heating Hybex the Set digHg-epBfe oHbxWtrahInsert Waterbath Hybex to Buffer High-Temp Adding ahBeadChip Wash npg 95 page on h etday. next the at#1081 e.D Rev. 15018210 # Part Wash BeadChip 95 Supplied By User Illumina Illumina Illumina Illumina temperature temperature temperature temperature temperature Quantity Storage BottleBottle Room Bottle Room Tube Room Bottle Room Room NOTE These procedures describe preparing 24 samplesDASL using HT a Assay 24 sample Profiling Reagent WG- Kit.DASL HT If Assay you Profiling are Reagent using Kit,to a the prepare 96 sample kit WG- 96 provides samples enough at reagent once. Each XC4 bottle contains enough to process up to 24 BeadChips. Add 335 ml 100% EtOH to the— XC4 bottle. The final volume is 350 ml. Item 100% EtOH High Temperature Wash Buffer PB1 Wash E1BC Buffer XC4 • In preparation for the CoatXC4 BeadChip protocol, reagent: follow these steps to resuspend the } Preparation Estimated Time Hands-on: 30 minutes Incubation: Two 5 minute washes, one 10 minute wash,Consumables one 1 hour incubation In this process, prepare forovernight the hybridization. wash Remove steps the by BeadChipBeadChips. removing coverseals the and BeadChips then from wash the the Whole-Genome Gene Expression DASL HT Assay Guide Wash BeadChip WG-DASL HT Assay Lab Protocols 96 eoeSeal Remove 2 1 } } lc fdltdWs 1Cbfe naPrxN.34 beaker. No. 3140 Pyrex a solution. in E1BC buffer Wash E1BC the Wash make diluted to of water RNase-free 1 L Place 2 L to buffer E1BC 6 ml Add umrete aeu ttebto ftebeaker. the of bottom and the Chamber Hyb at the up from face BeadChips them submerge all remove hands, gloved powder-free Using bench. lab the on it place chamber. and the oven the Disassemble from Chamber Hyb the Remove • • • vrih.Ke h C ntebtl nwihi a hpe ni ed for ready until shipped was it which 2° to 8°C in bottle at the it use. store in can XC4 the You temperature. Keep room overnight. at at vortex suspension. XC4 visible, complete use is in resuspended, coating Once is If in XC4 container. is the the XC4 until coating all still 1,625 rpm ensure is to none hand and by vigorously suspension bottle the frozen the shake to 30–40 minutes, opposite After side 30– the for on rocker possible. bottle a if the pellet on Place place resuspend. to and minutes 15 seconds, 40 for vigorously shake bottle, the Re-cap hme rmteoe,poesalo t edhp,ads nutlall until on so and BeadChips, its processed. of all second are process chambers remove oven, then the the below, in from 2–5 BeadChips steps all chamber in Process described time. oven as a the chamber at first from one them BeadChips the remove process chambers, and multiple processing are you If NOTE to Service beaker. not a Customer have Illumina obtain you contact Expression If please Gene Kit. recently, a Starter one of Universal purchased purchase or the Kit with Option comes Product (IVT) beaker 3140 No. Pyrex A NOTE at#1081 e.D Rev. 15018210 # Part Wash BeadChip 97 Removing the Coverseal BeadChips Submerged Face Up in Beaker Using tweezers or powder-free gloved hands,submerged transfer in the the BeadChip dish to containing the 250 ml slide rack Wash E1BC solution. Using powder-free gloved hands, remove theThis coverseal may from require the significant first force,the BeadChip. due entire to BeadChip the remains strength of submerged during the removal. adhesive.Figure Ensure 41 that Figure 40 4 3 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 98 ihTm Wash Temp High 2 1 5 iue42 Figure ls h ye lid. Hybex the insert Close Waterbath Hybex the into buffer. rack Wash the High-Temp transfer containing handle, rack slide the Using steps Repeat edhpi ahEB ufrbfr eoigtenx BeadChip Insert. next Chamber the Hyb removing its before from buffer E1BC each Wash submerge in BeadChips, BeadChip multiple Wash diluted processing the When in buffer. submerged E1BC completely is BeadChip the Ensure NOTE umrigBaCisi ahDs otiigEB Buffer E1BC Containing Dish Wash in BeadChips Submerging 3 and 4 o l edhp rmtesm y Chamber. Hyb same the from BeadChips all for at#1081 e.D Rev. 15018210 # Part Wash BeadChip 99 Transfer Wash Rack to Waterbath Insert Slide rack handle attached Transfer to Hybex Waterbath insert A B Incubate static for 10 minutes with the Hybex lid closed. Figure 43 3 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 100 is omTm Wash Room-Temp First 1 iue44 Figure rnfrtesierc akit ihcnann 5 lfehWs 1Cbuffer. E1BC Wash fresh 250 ml containing 45 dish Figure a immediately into complete, back is rack buffer slide Wash the High-Temp transfer in incubation 10 minute the After ttcIcbto nHg-epWs Buffer Wash High-Temp in Incubation Static ahn edhpi iue ahEB Buffer E1BC Wash Diluted in BeadChip Washing at#1081 e.D Rev. 15018210 # Part Wash BeadChip 101 Washing Dish/BeadChip on Orbital Shaker NOTE When processing multiple BeadChips, submergebuffer each before in the removing Wash the E1BC next BeadChip from its Hyb Chamber. Transfer the rack to a clean dishUsing containing the slide 250 ml rack fresh handle, 100% Ethanol. plunge thePlace rack the in dish and on out the of orbital the shaker solution and 5–10 shake times. at room temperature for 10 minutes. Using the slide rack handle, plunge theSet rack the in orbital shaker and to out medium-low. of thePlace solution the 5–10 times. dish on theShake orbital at shaker as and high shake athe at dish. speed room as temperature for possible 5 minutes. without allowing theFigure solution 46 to splash out of 1 2 3 2 3 4 Whole-Genome Gene Expression DASL HT Assay Guide Ethanol Wash WG-DASL HT Assay Lab Protocols 102 otBeadChip Coat 3 2 1 notes: important these read please process, BeadChip Coat the Racks starting Before Tube and Dishes Wash Prepare e ptotplaigws ihs aee sP1adXC4. and PB1 as labeled dishes, wash top-loading two up Set necessary, If dissolved. resuspension. completely complete ensure until to vortex vigorously bottle XC4 the Shake bench: lab the on equipment following the out Lay • • • • • • • • • • aumhose Vacuum Kimwipes Large tweezers Self-locking rack tube 1 desiccator vacuum 1 rack staining 1 rack. tube this per under required rack are one the Allow of Kimwipes BeadChips. No dimensions the internal 8 BeadChips. of the removal fits for that desiccator rack vacuum tube clean the additional from it an Prepare removing after the rack the dish. tube place of wash this will top XC4 on You on diapers. BeadChips rack tube lab containing absorbent a rack dry. on Place staining to place bench. rack not lab Do wash the layers. a on Kimwipe on layers three down in upside Kimwipes dishes Place wash following Immediately and water. racks dishes DI place wash with wash, and Rinse racks use. tube after and wash before coating, thoroughly to BeadChip air XC4 low-pressure for with preparation dishes In use. wash wash to on Clean prior use. covers particulates in dish wash remove not the wash entering or Place stored dust BeadChips. when or the lint dishes to of transfer chance could the which minimize dishes, to care utmost the Take at#1081 e.D Rev. 15018210 # Part Wash BeadChip 103 . This orients the staining rack so that you can safely remove the PB1 and XC4 Wash Dishes with Staining Rack Wash Dishes Staining Rack A B facing you To indicate the fill volume310 ml before filling water wash into dishes the washwater with from PB1 dishes the and and wash XC4, mark dish. pour and the This water XC4 enables level bottles you on into to the thelabware pour side. wash to reagent Empty dishes, directly wash the from minimizing dishes. the contaminant PB1 transferPour from 310 ml PB1 into the washSubmerge dish the unloaded labeled “PB1.” staining racktab into the wash dishBeadChips. with the locking arms and Figure 47 4 5 6 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 104 8 7 iue48 Figure fncsay rel ittesann akoto h ahds osa h BeadChip. the seat to dish BeadChip. the wash the inserting after of immediately out it rack Replace staining the lift briefly barcodes necessary, BeadChip four If The Put below. PB1. four in and submerged handle is should rack it staining while the rack above staining BeadChips the in BeadChips the Place is it while rack BeadChips. staining the the carry to to it PB1. wash EtOH use in the submerged will from You BeadChip dish. each wash transfer Quickly the in sit rack staining the Let B A aeaway face Tab Arms Locking onttuhtefc fteBaCis adete ytebarcode the by edges. them Handle the BeadChips. by the or of end face the touch not Do CAUTION before handle rack staining BeadChips. may the the BeadChips removing remove the you oriented, when correctly damaged not is be handle rack staining the If CAUTION tiigRc okn rsadTab and Arms Locking Rack Staining rmyu hl h okn rso h handle the on arms locking the while you, from at#1081 e.D Rev. 15018210 # Part aetowards face you. Wash BeadChip 105 face away you, for proper face towards Washing BeadChips in PB1 NOTE Use the XC4 within 10 minutes after filling the wash dish. NOTE If the top edges ofXC4 the washes, BeadChips gently begin move tothe the touch slides. staining during It rack either is back PB1 important or andBeadChips. for forth the to solution separate to circulate freely between all CAUTION Do not leave the BeadChips submerged in PB1 for longer than 30 minutes. CAUTION Do not allow the BeadChipsas to soon dry. as Submerge possible. each BeadChip in the wash dish from you, while the lockinghandling arms and on the coating. handle Place the bottle with excessXC4 XC4 wash in dish a during readily the available coating locationRemove procedure. for the topping staining off the rack fromwash the dish dish containing containing XC4. PB1 The and barcode place labels it on directly the into BeadChips the must Allow the BeadChips to soak for an additional 5 minutes. Pour 310 ml XC4 intolint the dish or dust labeled from “XC4,” falling and into cover the the solution. dish to prevent any Move the staining rack up andFigure down 49 10 times, breaking the surface of the PB1. 12 13 10 11 9 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 106 18 17 16 15 14 iue50 Figure eoetesann aki n moh ai oinadpaei ietyo the on directly it place barcodes down and face the motion sure rapid making smooth, rack, one tube prepared in rack staining the dimensions Remove internal the fits that desiccator. BeadChips vacuum 8 the per of rack tube additional Kimwipes folded one two Prepare placing by rack rack. staining tube the the for under rack tube clean a Prepare 5 minutes. additional an for soak to BeadChips the Allow XC4. the of surface the breaking 10 times, down and up rack staining the Move s C nyoc.T rcs usqetBaCis s e,clean new, a use XC4. BeadChips, fresh subsequent with dish process wash To once. only XC4 Use CAUTION h lds ti motn o h ouint iclt reybtenall between freely circulate to separate solution to the forth for BeadChips. and or important PB1 back is either rack It during staining slides. touch the the to move begin gently BeadChips washes, the XC4 of edges top the If NOTE oigBaCisfo B oXC4 to PB1 from BeadChips Moving . aeup face n h okn rsadtab and arms locking the and at#1081 e.D Rev. 15018210 # Part Wash BeadChip 107 Moving the Staining Rack from XC4 to Tube Rack Staining Rack in Correct Orientation Continuing to hold the stainingbarcode rack end handle, with carefully self-locking grip tweezers. each BeadChip at its For the top four BeadChips, workinga top to bottom: To ensure uniform coating, placeavoiding the the staining raised rack edges. on the center ofFigure the 52 tube rack, Figure 51 20 19 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 108 22 21 akadtoBaCiso h otm h acd nssol etwrsyou, towards be horizontal. completely should the be ends of barcode should top The BeadChips on bottom. the BeadChips the and six on with BeadChips rack two tube and the rack to BeadChips remaining the Remove remove. away and handle handle 53 the the Figure push up and Pull thumb your it. your between unlock with handle to up the you tab from grasp the position, Push in forefinger. rack and staining thumb the of top the Holding b lc h edhpo uerc ihtebroefcn padtwrsyou. towards and up facing barcode the with rack tube a on BeadChip the Place B A Handle Tab eflcigtezr rpteBaCi imyadhl prevent help The and hold. firmly damage. to BeadChip difficult BeadChips the the grip makes tweezers and self-locking slippery is coat XC4 The NOTE eoigSann akHandle Rack Staining Removing at#1081 e.D Rev. 15018210 # Part Wash BeadChip 109 Testing Vacuum Seal Placing BeadChips on Tube Rack Dry under vacuum for 50–55Drying minutes. times may vary according to room temperature and humidity. To prevent wicking and unevenedge drying, of do the not tube allow rack or the to BeadChips touch toPlace each rest the on other tube while rack the drying. inrack the (8 vacuum BeadChips). desiccator. Each dessicator canEnsure hold the one vacuum tube valve is seatedRemove tightly the and red securely. plug from the three-way valveStart the before applying vacuum, vacuum using pressure. at leastTo 508 ensure mm that Hg the (0.68 dessicatordesiccator. bar). is It properly should sealed, not gently lift lift off the the lid desiccatorFigure of base. 55 the vacuum Figure 54 28 23 24 25 26 27 Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT Assay Lab Protocols 110 29 35 34 33 32 31 30 ees h aumb unn h adevr slowly. very handle the turning by vacuum the Release icr nsdraet nacrac ihfclt standards. to Proceed facility with accordance in reagents unused Discard c b a Chambers: Hyb the Clean c b have to likely most a the are to BeadChips BeadChip two excess. bottom the some The of underside XC4. the excess clean any remove manually tacky, feels touch. underside the the to If dry are BeadChips the of ( side bar-coded chips the from of plug borders red the the Touch Remove pressure. port. vacuum valve to applying the before valve within three-way valve three-way lint desiccator’s the the and in dust of plug accumulation valve red stop the with desiccator the Store hruhyrneteegthmdfigbfe reservoirs. buffer humidifying eight water. the DI rinse with Thoroughly components Chamber Hyb Chambers. all Hyb Rinse the from gaskets rubber the Remove is surface the until times, six or smooth. five and BeadChip the clean of finger. underside index the your along around Wipe Wipe EtOH Prostat dripping pre-saturated from a EtOH Wrap excess prevent to stripes. angle the downward onto a at BeadChip the Hold onot Do CAUTION the desiccator. see instructions, while the plug desiccator with included vacuum valve documentation detailed the For applied. remove to is you vacuum damage if in a true result especially can is desiccator This vacuum BeadChips. the the the disturbing avoid of use to slowly Improper very contents. desiccator the enter should Air WARNING mg BeadChip Image oc h stripes. the touch npg 111 page on onttuhtestripes the touch not do . oesr htteetched, the that ensure to ) at#1081 e.D Rev. 15018210 # Part Image BeadChip 111 iScan System , for general instructions on scanning your Direct Hyb Scan Setting BeadArray Reader User Guide DirectHyb Gene Expression HiScanSQ System User Guide WG-DASL HT Assay Scan Settings , or the Scanner iScan or HiScan BeadArray Reader Table 15 Proceed to scanning the BeadChips.User See Guide the BeadChips. For specific scan settings refer to the following table: Whole-Genome Gene Expression DASL HT Assay Guide Image BeadChip WG-DASL HT Assay Lab Protocols 112 GenomeStudio ud n h eoetdoGn xrsinMdl srGuide. User Module User Expression Framework Gene GenomeStudio the GenomeStudio see the data, to and expression platform Guide gene GenomeStudio the analyze using and on visualize instructions and the Reader. descriptions BeadArray feature as or For such System systems, iScan from or collected HiScan images gene microarray Illumina analyzing scanned for from application data an expression is Module Expression GenomeStudioGene The at#1081 e.D Rev. 15018210 # Part Appendix A 114 115 116 117 118 119 113 View the Control Report Negative Controls Oligo Annealing Controls Array Hybridization Controls Gene Intensity (Housekeeping and All Genes) Introduction Appendix A WG-DASL HT AssayControls WG-DASL HT AssayControls Whole-Genome Gene Expression DASL HT Assay Guide WG-DASL HT AssayControls 114 Introduction hsapni ecie h oto lgsfrteW-ALH sa n o to how and Assay include: HT oligos WG-DASL control the The for them. oligos view control the describes appendix This } } } } eeItniy(oskeigadAlGenes) All and (Housekeeping Intensity Gene Controls Hybridization Array Controls Annealing Oligo Controls Negative npg 116 page on npg 117 page on npg 118 page on npg 119 page on at#1081 e.D Rev. 15018210 # Part View the Control Report 115 GenomeStudio Framework User Guide and the GenomeStudio The GenomeStudio software platform tracksgenerates the a performance of report across these controls all and arraysFor on more the information, BeadChip. see the Gene Expression Module User Guide. Whole-Genome Gene Expression DASL HT Assay Guide View the Control Report WG-DASL HT AssayControls 116 eaieControls Negative hs rbst sals eeepeso eeto limits. of detection deviation expression standard gene The signal establish dye. to of and probes binding signals these non-specific the or uses cross-hybridization platform from software background system resulting GenomeStudio system the signal defines imaging any probes the by these both of by and represented not signal is do mean background that The This sequences genome. background. random human the ~300 targeting in oligos appear query of consists category This iue56 Figure eaieCnrlDiagram Control Negative at#1081 e.D Rev. 15018210 # Part Oligo Annealing Controls s to m 117 ASO should give higher signals than m Oligo Annealing Controls ASO. m Figure 57 The oligo annealing controls testthe the same efficiency cDNA of target. annealing Inthe ASOs each lower with case, T different the T higher T Whole-Genome Gene Expression DASL HT Assay Guide Oligo Annealing Controls WG-DASL HT AssayControls 118 o tignyHbControl Hyb Stringency Low Controls Hyb Cy3-Labeled Controls Hybridization Array inl fsrnec stolw hyyedsga prahn hto hi efc match perfect their of that category. control approaching Hyb signal Cy3 low yield the they very in low, yield bases counterparts too controls mismatch these is adequate, two stringency If is has stringency probe signal. If each sequence. case, its high- this in and In distributed medium- targets. the control to Hyb corresponding Cy3 probes, concentration four contains category control This responses. hybridization concentrations gradient three reactions. yielding at prep high), present sample and are the medium, controls of (low, Hyb success Cy3 the and for quality signal oligonucleotides RNA Target a cellular produce they the both hybridization, of successful oligonucleotidesindependent Following Cy3-labeled HYB. corresponding the with probes in present six of consist controls These category: this dye comprise Cy3 controls of with types labeled Two oligos 50-mer reagent. of HYB consist the controls in The included assay beads. single-stranded array of the hybridization to the products test controls hybridization array The } } iue59 Figure 58 Figure Control Hyb Stringency Low Control Hyb Cy3-Labeled o tignyHbCnrlDiagram Control Hyb Stringency Low Diagram Control Hyb Cy3-Labeled at#1081 e.D Rev. 15018210 # Part Gene Intensity (Housekeeping and All Genes) 119 WG-DASL HT Control Summary Figure 60 The intactness of the biologicalcontrols. specimen These controls can consist be monitored ofexpressed by probes in housekeeping to most gene housekeeping samples. genes that typically are Whole-Genome Gene Expression DASL HT Assay Guide Gene Intensity (Housekeeping and All Genes) 120 Part# 15018210 Rev. D Appendix B 122 123 126 121 Make Single-Use DNA (SUD) Plate Data Analysis Introduction Appendix B Assay Qualification Using gDNA Assay Qualification Using gDNA Whole-Genome Gene Expression DASL HT Assay Guide Assay Qualification Using gDNA 122 Introduction sasdrn aaaayi nteGnmSui otaeplatform. target software GenomeStudio qualify individual the to in used be analysis then data sample. Single-Use can during control (Make samples SUD positive assays gDNA Make a the the as from use derived include can to The data gDNA user boundaries. activated exon to prepare oligos Assay within process the HT DNA) fall WG-DASL because to the possible designed option, is are an This As target sample. Assay each a HT of WG-DASL performance as the the DNA in that genomic used is using tested be Assay can HT WG-DASL site the of feature unique One at#1081 e.D Rev. 15018210 # Part Make Single-Use DNA (SUD) Plate in the 123 Supplied By Illumina User User once Sample Sheet -15° to -25°C See manufacturer’s instructions Quantity Storage Seeinstructions Roomtemperature1 tube per SUD plate 50 ng/µl User plate -15° to -25°C . Date and time Operator Item 10 mM Tris-HCl pH 8.0,EDTA 1 (TE) mM MS1 reagent Human genomic DNA 96-well 0.2 ml skirted microplate 1 per sample • • on page 31 Preheat the heat block to 95°CTurn and on allow the heat the sealer temperature toThaw to stabilize. preheat the it. MS1 Allow reagent 15 minutes. tubepour to the room entire temperature. Vortex tube into to mix aThaw new, the the non-sterile contents, DNA reservoir. and samples andcontents. controls to room temperature andApply vortex a to SUD mix barcode the label toOn a the lab new 96-well tracking form, microplate. record: In the appropriate columns ofSample_Plate the for Sample each Sheet, Sample_Well enter the defined Sample_Name in the and Sample Sheet. See } } } } } } } Preparation This process activates sufficientWG-DASL DNA HT of Assay. each individual sample to be used Estimated Time Hands-on: ~15 minutes Incubation: 30 minutes Consumables Whole-Genome Gene Expression DASL HT Assay Guide Make Single-Use DNA (SUD) Plate Assay Qualification Using gDNA 124 aeSUD Make 11 10 9 8 7 6 5 4 3 2 1 us etiuetepaet 250 xg. form. to tracking plate the lab centrifuge the Pulse on times stop and start the Record seal. cover, plate block the heat on the condensation Using reduce minutes. to 30 plate exactly SUD for the lid. 95°C cover the at close plate and SUD block the heat Incubate preheated the in plate SUD the Place the 250 xg. to to centrifuge strapped firmly Pulse is movement. plate plate the prevent sure to making platform vortexer 20 seconds, for 2300 rpm at Vortex (3 250 xg. sealer to heat plate the SUD with the it centrifuge sealed. seal Pulse completely and are plate wells SUD all the that to Ensure seal seconds). heat foil microplate of well a dispenses. Apply each column to between sample tips DNA Change normalized plate. SUD 5 µl the transfer plate. pipette, SUD 8-channel the an of well Using EDTA. each to 1 mM reagent pH 8.0, MS1 Tris-HCl 5 µl 10 mM Add with 50 ng/µl to samples DNA Normalize • • S egn barcode reagent MS1 barcode plate SUD ontalwte9° nuainpro oece 30 minutes. exceed to period incubation 95°C the allow not Do CAUTION nuaint rvn h el rmdyn u uigincubation. 95°C during the out before drying xg from 250 wells to the plate prevent SUD to the incubation centrifuge to important is It NOTE to Go hand. by in tracking filled form. lab and form printed the This download or form. online, to http://www.illumina.com/documentation tracking saved lab and out the filled use information, be barcodes, can and operator as times, such stop assay and your start about information record To NOTE at#1081 e.D Rev. 15018210 # Part Make Single-Use DNA (SUD) Plate 125 If you plan to performblock the to Make 70°C. ASE protocol today, then immediately setTo use the heat the activated gDNASUD in product the in Make place ASE of protocol,ASE the substitute plate usual 10 µl will 10 µl of contain the of 10 µl Make the DAP, Make 30 µl OB1, SUR product. and Each 10 µl well of of the either SUR or SUD. 12 13 Whole-Genome Gene Expression DASL HT Assay Guide Assay Qualification Using gDNA 126 aaAnalysis Data ape ntesm a sfrtecN ape.Adsrpino o hsdt is data this how of the description in A provided samples. is cDNA Guide probes the suboptimal for eliminate gDNA as to the way used from same data the intensity in array extract samples will platform software GenomeStudio The . eoetdoFaeokUser Framework GenomeStudio at#1081 e.D Rev. 15018210 # Part Index 127 cap mats 25 PCR product 25 pipetting technique 67 seal removal tipsUDG 74 26 Gene Expression 114 amplification contamination 25 cDNA Synth MCS4 Kitcentrifuge, balance 40 30 cleaning procedures 22 configuration 34 contamination control oligos 56 customer support 131 cycle PCR 76 D DAP 17, 31-32,DASL 39, 63 17 data analysis 13,Deoxyribonucleic Acid 112 (DNA) 17 DEPC 17 differential analysis 13,dispense 112 speed 81 DNA 17 DNA polymerase 27 DNA Polymerase 40 documentation 131 Downstream-Specific Oligo 17 DSO 17 dUTP 26 Gene Expression 118 equipment 34 optional stopping pointsUDG, 62, with 76 or withoutworkflow 26 6 labels 25 pipettes 30 handling techniques 25 A acronyms 17 AM1 17, 39 amplification contamination 25 array hybridization controls ASE plate 17,ASO 39, 63 17 assay Assay-specific oligos 63 Assay Specific Oligo (ASO)AutoLoader 17 12 AutoLoader2 12 B balance centrifuge 30 barcodes BeadArray Reader 12, 112 best practices 25 bind PCR products 77bleaching procedures 22 C calibrate cap mats 25 cDNA 17 Index Whole-Genome Gene Expression DASL HT Assay Guide Index 128 Sa ytm1,1,3,112 34, 12, 10, System 72 iScan 39, 18, IP1 45 39 Invitrogen 17, plate INT 34 guide product Illumina I 17 Hybridize 17 Buffer Hybridization 17 Hybridization 39 17, HYB 17 Hyb 39 17, HTW 112 41 12, 4, HiScan Kit Paraffin RNA Pure High 131 technical help, 35 sealer heat 39 17, HCB H 4 guanidine 112 13, GenomeStudio 12 112 GenomeScan 13, analysis gene G 24 warning formamide 4 formamide 31 tracking lab form, 35 fluorometer 72 primers labelled fluorescently F 66 ligation and extension 17 EtOH 17 Ethanol 34 equipment 39 17, E1BC E C lt 8 9 70 39, 18, plate PCR PCR 39 18, 76 PB1 62, points 77 pause 63, particles paramagnetic P 66 excess or mis-hybridized oligos, controls annealing 63 oligo 39, 18, OB1 O controls negative 39 7, RefSeq NCBI 18 NaOH N 24 formamide for 77 MSDS 39, 18, MPB 39 18, 66 MH1 39, 60 18, 56, 39, MEL 18, iii, MCS4 iii MCS3 M 31 form tracking lab L 34 options kit K hra ylr3,76 35, cycler thermal 25 contamination product 72 primers 63 20 site primer areas post-PCR and pre- 77 products bind 117 Expression Gene 116 Expression Gene at#1081 e.D Rev. 15018210 # Part Index 129 70 input amount 6 number of 26 overview 4 safety in lab proceduresSample 16 QRNA platesample 45 sheet 31 samples SCM 18,39,70SDS 18, 28 signal intensity 10 Single-Use RNA (SUR) PlateSodium 60 Hydroxide (NaOH) 18 spectrofluorometer 35 Standard QRNA plate 45stop, optional 62,SUR 76 18, 39 T technical assistance 131 thermal cycler 35,Titanium 76 Taq 26-27, 40 U UB1 18,40,72UB2 18, 40 UDG 19, 40 UHR Total RNAUniversal 59 Starter KitUracil 34 DNA Glycosylase (UDG) 26, USO 19 V vortexer 29 W workflow 6 X XC4 solution 19,xg 40 19 clean and calibratecontamination 30 prevention 67 dispense technique 26 cap mats 25 RNA 18 dispense technique 26 names 17 reuse 25 quantification 18 ribosomal RNA 45 samples 4 cap mats, handlingcontamination 25 24 formamide 24 government standards 24 pipettes plate labels 25 plates PMPs 18 PS1 18, 39 Q QRNA 18, 39 Quant-iT Assay Kitquantification 41, 45 quantitate RNA 45 R reagents RiboGreen 41, 45 RiboGreen RNA quantitation kitRibonuclease 45 (RNase) 18 Ribonucleic Acid (RNA) 18 RNA 18 RNA Paraffin KitRNase 4, 41 18 RNase-Free techniques 28 RTE iii, 18, 39 S safety Whole-Genome Gene Expression DASL HT Assay Guide Index 130 at#1081 e.D Rev. 15018210 # Part Technical Assistance 131 Contact Number 800.874909 0800.0223859 800.16836 900.812168 020790181 0800.563118 Italy Netherlands Norway Spain Sweden Switzerland United KingdomOther countries 0800.917.0041 +44.1799.534000 http://www.illumina.com [email protected] Contact Number Region 0800.296575 0800.81102 80882346 0800.918363 0800.911850 0800.180.8994 1.800.812949 Email Illumina General Contact Information Illumina Customer Support Telephone Numbers Illumina Website Region North AmericaAustria 1.800.809.4566 Belgium Denmark Finland France Germany Ireland MSDSs Material safety data sheets (MSDSs)http://www.illumina.com/msds. are available on the Illumina website at Product Documentation You can obtain PDFs ofGo additional to http://www.illumina.com/support product and documentation select fromdocumentation, a the you Illumina product. will To website. be download askedview to or log save in the to PDF.https://my.illumina.com/Account/Register. MyIllumina. To After register you for log a in, MyIllumina you account, can please visit Table 16 Table 17 Technical Assistance For technical assistance, contact Illumina Customer Support. Whole-Genome Gene Expression DASL HT Assay Guide Illumina Headquartered in San Diego, California, U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) [email protected] www.illumina.com