GeneChip TM HT WT User Guide

Transcription

1 GeneChip TM HT WT User Guide User Manual For Research Use Only. Not for use in diagnostic procedures.

2 Information in this document is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Corporate entity Life Technologies Carlsbad, CA USA Toll free in USA Trademarks All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. All other trademarks are the property of their respective owners Thermo Fisher Scientific Inc. All rights reserved. P/N

3 Contents Chapter 1 About the HT and Ambion TM WT Expression Assay... 5 Chapter 2 RNA Preparation Quantitation of RNA Preparation of Poly-A RNA Controls Preparation of Total RNA Plates for Processing on Automated Systems Chapter 3 Labware and Reagents Required Equipment, Consumables, Labware and Reagents Required Equipment and Labware Required Reagents Required Labware Labware Templates Reagent & Labware Requirements (Part 1 to Part 6) Reagent and Labware Required for Part Reagent and Labware Required for Part Reagent and Labware Required for Part Reagent and Labware Required for Part Reagent and Labware Required for Part Reagent and Labware Required for Part Chapter 4 Beckman Coulter Biomek TM FX P Express Setup and Target Preparation Part 1 - First and Second-Strand cdna Synthesis, IVT crna Synthesis Approximate Duration Reagents and Labware Required Clean the Bio-Rad 96-Well Hard-Shell PCR Plate Lids Prepare the Biomek FX P TPE System Breaking the Light Curtain While Running the Assay Thaw and Prepare the Samples and Reagents Start the Biomek Software Begin Part 1: First and Second-Strand cdna Synthesis, IVT crna Synthesis Part2-cRNAPurification Approximate Duration Reagents and Labware Required Perform a Pre-run Check of the Biomek FX P TPE System Prepare the Samples and Reagents Continue the Run - Part 2: crna Purification Part 3 - Second Cycle cdna Synthesis & RNase H Hydrolysis Approximate Duration GeneChip TM HT WT User Guide

4 Perform a Pre-run Check of the Biomek FX P TPE System Continue the Run - Part 3: Second Cycle cdna Synthesis & RNase Hydrolysis Part 4 - cdna Purification Approximate Duration Reagents and Labware Required Perform a Pre-run Check of the Biomek FX P TPE System Thaw and Prepare the Samples and Reagents Continue the Run - Part 4: cdna Purification Part 5 - cdna Fragmentation, Labeling, & Hybridization Setup Approximate Duration Reagents and Labware Required Perform a Pre-run Check of the Biomek FXP TPE System Thaw and Prepare the Samples and Reagents Continue the Run - Part 5: cdna Fragmentation, Labeling & Hybridization Setup Part 6 - GeneTitan TM Instrument Hybridization Preparation Approximate Duration Reagents and Labware Required Perform a Pre-run Check of the Biomek FX P TPE System Labeling GeneTitan Reagent Trays Thaw and Prepare the Samples and Reagents Anti-Static Procedure for GeneTitan Instrument Trays and Covers Continue the Run - Part 6: Preparation for the Gene Titan Instrument Appendix A Using the AmbionTM 24 Rxn Kit for Two 8-Sample Runs Appendix B GeneChipTM HT WT Workflow Diagram First Strand cdna Synthesis Second Strand cdna Synthesis In Vitro crna Synthesis crna Purification (diagram 1 of 8) crna Purification (diagram 2 of 8) crna Purification (diagram 3 of 8) crna Purification (diagram 4 of 8) crna Purification (diagram 5 of 8) crna Purification (diagram 6 of 8) crna Purification (diagram 7 of 8) crna Purification (diagram 8 of 8) Second Cycle cdna Synthesis Denaturation Second Cycle cdna Synthesis - Synthesis GeneChip TM HT WT User Guide 3

5 RNase H Hydrolysis cdna Purification (diagram 1 of 8) cdna Purification (diagram 2 of 8) cdna Purification (diagram 3 of 8) cdna Purification (diagram 4 of 8) cdna Purification (diagram 5 of 8) cdna Purification (diagram 6 of 8) cdna Purification (diagram 7 of 8) cdna Purification (diagram 8 of 8) Fragmentation Labeling Cartridge Hyb Setup (diagram 1 of 2) Cartridge Hyb Setup (diagram 2 of 2) Array Plate Hyb Setup (diagram 1 of 2) Array Plate Hyb Setup (diagram 2 of 2) GeneTitan Instrument Preparation (diagram 1 of 2) GeneTitan Instrument Preparation (diagram 2 of 2) GeneChip TM HT WT User Guide

6 Chapter 1 About the HT and Ambion TM WT Expression Assay The High Throughput and Ambion TM WT Expression Assay uses the Ambion TM WT Expression Kit in conjunction with the GeneChip TM HT WT Terminal Labeling Kit to generate amplified and biotinylated sense-strand DNA targets from the entire expressed genome without bias. The assay and the associated reagents are for use with GeneChip TM ST arrays or GeneChip TM ST 1.1 array plates where ST stand for Sense Target. The probes on these arrays have been selected to be distributed throughout the entire length of each transcript. NOTE: The HT and Ambion TM WT Expression Assay is not compatible with GeneChipTM brand arrays designed to focus on the 3 ends of transcripts. For the 3 arrays, please use the GeneChip TM HT 3 IVT Express Kit The Ambion WT Expression Kit for high throughput robotics enables you to prepare RNA samples for whole transcriptome (WT) analysis. The kit generates sense-strand cdna from total RNA for subsequent fragmentation and labeling using the GeneChip TM HT WT Terminal Labeling Kit. It is optimized for use with human, mouse, or rat GeneChip Sense Target (ST) Arrays or Array Plates. The Ambion WT Expression Kit represents the latest innovation in RNA amplification that enables faster, more sensitive, and highly reproducible results with whole transcriptome microarrays and array plates. This kit features: A streamlined workflow that requires as little as 50 ng of total RNA for a single round of amplification Direct amplification of total RNA without a separate rrna depletion step A novel priming method that selectively reverse transcribes non-ribosomal RNA for complete and unbiased coverage of the transcriptome Magnetic-bead crna purification for high recovery and ease of use Pre-formulated master mixes that significantly reduce hands-on time The kit is based on a novel reverse transcription (TRT) priming method that eliminates the need for a separate rrna depletion step. The kit includes RT primers designed with a proprietary oligonucleotide matching algorithm that eliminates primer sequences with homology to known ribosomal RNA sequences. The result is complete and unbiased coverage of the transcriptome, with a significant reduction in rrna amplification, compared to other methods. The High Throughput and Ambion TM WT Expression Assay is developed for the Beckman Coulter Biomek TM FX P Target Prep Express (TPE) System. This system uses robotic technology to automate many of the labor intensive tasks required when preparing eukaryotic RNA for whole transcriptome expression analysis. Figure 1.1 and Figure 1.2 shows the workflow of the High Throughput and Ambion TM WT Expression Assay. NOTE: We do not recommend the use of user-prepared reagents for the HT WT Express Assay. Please use the GeneTitan TM Hybridization, Wash and Stain Kit for WT Array Plates (array plates, P/N ) or the GeneChip TM Hybridization, Wash and Stain Kit (cartridge arrays, P/N ) for all the reagents needed for the hybridization, wash and stain steps of this assay. The Beckman Coulter Biomek FX P TPE System automates the preparation of target that can be hybridized to a: GeneChip TM Cartridge Array that can be processed using the Hybridization Oven 640/645, GeneChip Fluidics Station FS450, and GCS 3000 Scanner System Target preparation for cartridge arrays includes five parts with an optional stopping point at the completion of each part (Figure 1.1). At the end of target preparation, the sample is ready to be denatured and hybridized onto a GeneChip ST Cartridge Array. GeneChip TM HT WT User Guide 5

7 GeneChip TM HT Array Plate and processed using the Gene Titan Instrument Target preparation for array plates includes six parts for array plates (Figure 1.2) with an optional stopping point at the completion of each part. At the end of target preparation, the denatured sample is ready to be fragmented and hybridized onto a GeneChip ST Array Plate. Table 1.1 to Table 1.6 provide the time required to perform each part of the assay. Depending on the selected parameters, target preparation takes about 30 hours and six human user interventions. Additional time is required for hybridization, washing, staining, and scanning, and is dependent on the number of samples processed and the array type. NOTE: Complementary RNA (crna) is also known as amplified RNA (arna). This manual describes the assay procedures recommended for eukaryotic target labeling for whole transcriptome expression analysis using the Beckman Coulter Biomek TM FX P TPE System. The reagents and protocols have been developed and optimized specifically for use with the Beckman Coulter Biomek TM FX P TPE System. It is anticipated that by following the recommended procedures, you will be able to generate sufficient biotin-labeled complementary DNA (cdna target from each sample (8, 16, 24 or 96 sample workflow) for hybridization to a single GeneChip TM Cartridge or Array Plate. For information on RNA preparation, please see the Chapter 2, RNA Preparation. 6 GeneChip TM HT WT User Guide

8 Figure 1.1 Overview of the Ambion High Throughput WT Expression Assay - Cartridge Arrays GeneChip TM HT WT User Guide 7

9 Figure 1.2 Overview of the Ambion High Throughput WT Expression Assay - Array Plates 8 GeneChip TM HT WT User Guide

10 Table 1.1 Approximate Time Required for Part 1 Part 1 - First & Second-Strand cdna Synthesis, IVT crna Synthesis 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: First strand synthesis Thermal cycler first strand method Biomek TM FX P TPE System pipetting steps: Second strand synthesis Thermal cycler second strand method Biomek TM FX P TPE System pipetting steps: IVT Thermal cycler: IVT method Part 1 Total Time 1180 min ~19.7 hr 1182 min 19.7 hr 1185 min hr 1218 min 20.3 hr Table 1.2 Approximate Time Required for Part 2 Part 2 - crna Purification 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: crna purification Normalization Part 2 Total Time 81 min 1.35 hr 82 min 1.37 hr 83 min 1.38 hr 109 min 1.82 hr Table 1.3 Approximate Time Required for Part 3 Part 3 -Second Cycle cdna Synthesis 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: Second cycle denaturation Thermal cycler denaturation method Biomek TM FX P TPE System pipetting steps: Second cycle synthesis Thermal cycler synthesis method Biomek TM FX P TPE System pipetting steps: RNaseH hydrolysis Thermal cycler: RNaseH method Part 3 Total Time 195 min 3.25 hr 197 min 3.28 hr 197 min 3.28 hr 218 min 3.63 hr GeneChip TM HT WT User Guide 9

11 Table 1.4 Approximate Time Required for Part 4 Part 4 - cdna Purification 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: cdna purification Normalization Part 4 Total Time 84 min 1.40 hr 84 min 1.40 hr 84 min 1.40 hr 108 min 1.80 hr Table 1.5 Approximate Time Required for Part 5 Part 5 - cdna Fragmentation, Labeling, and Hybridization Setup 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: cdna fragmentation Thermal cycler fragmentation method Biomek TM FX P TPE System pipetting steps: Labeling Thermal cycler labeling method Biomek TM FX P TPE System pipetting steps: Hybridization setup GeneChip TM Cartridge Array Hybridization setup Or or or or or GeneChip TM Array Plate Hybridization setup N/A Part 5 Total Time - Cartridge Array 164 min 2.73 hr 167 min 2.78 hr Part 5 Total Time - Array Plate N/A 168 min 2.80 hr 167 min 2.78 hr 168 min 2.80 hr 185 min 3.08 hr 186 min 3.10 hr Table 1.6 Approximate Time Required for Part 6 Part 6 - GeneTitan Hyb Prep 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup N/A Biomek TM FX P TPE System pipetting steps N/A Thermal cycler: denaturation method N/A Part 6 Total Time N/A 32 min 52 min 82 min 1.37 hr 10 GeneChip TM HT WT User Guide

12 Chapter 2 RNA Preparation This chapter describes the general requirements for starting total RNA sample, poly-a control preparation for spiking into your RNA sample, and preparation of total RNA plates for processing on automated systems. IMPORTANT:. The quality of the RNA is essential to the overall success of the analysis. Since the most appropriate protocol for the isolation of RNA can be source dependent, we recommend using a protocol that has been established for the tissues or cells being used. In the absence of an established protocol, using one of the commercially available kits designed for RNA isolation is suggested When using a commercial kit, follow the manufacturer s instructions for RNA isolation. Quantitation of RNA Quantify RNA yield by spectrophotometric analysis using the convention that 1 absorbance unit at 260 nm equals 40 µg/ml RNA. The absorbance should be checked at 260 and 280 nm for determination of sample concentration and purity. The A 260 /A 280 ratio should be close to 2.0 for pure RNA (ratios between 1.7 and 2.1 are acceptable). Integrity of total RNA samples can also be assessed qualitatively on an Agilent 2100 Bioanalyzer. Refer to Figure 2.1 for an example of good-quality total RNA sample. Figure 2.1 Electropherogram (from the Agilent 2100 Bioanalyzer) for HeLa Total RNA. For a high-quality total RNA sample, two well-defined peaks corresponding to the 18S and 28S ribosomal RNAs should be observed, similar to a denaturing agarose gel, with ratios approaching 2:1 for the 28S to 18S bands. GeneChip TM HT WT User Guide 11

13 Preparation of Poly-A RNA Controls Reagents and Equipment Ambion TM WT Expression Kit for High Throughput Robotics: Ambion P/N (1 x 24 rxn) or P/N (1 x 96 rxn). GeneChip TM HT WT Terminal Labeling and Control Kit: P/N (24 rxn) or P/N (96 rxn) Bundle Includes: GeneChip TM HT WT Terminal Labeling Kit GeneChip TM Hybridization Control Kit GeneChip TM Poly-A RNA Control Kit Components used in this step: Eukaryotic Poly-A RNA Controls Designed specifically to provide exogenous positive controls to monitor the entire eukaryotic target labeling process, a set of poly-a RNA controls is supplied in the GeneChip TM Poly-A RNA Control Kit. Each eukaryotic GeneChip probe array contains probe sets for several B. subtilis genes that are absent in eukaryotic samples (lys, phe, thr, and dap). These poly-a RNA controls are in vitro synthesized, and the polyadenylated transcripts for the B. subtilis genes are pre-mixed at staggered concentrations. The concentrated Poly-A Control Stock can be diluted with the Poly-A Control Dil Buffer and spiked directly into RNA samples to achieve the final dilutions (relative to estimated copy number of total mrna population) summarized in Table 2.1. Table 2.1 Final Dilutions of Poly-A RNA Controls in Samples Poly-A RNA Spike Final Dilution (estimated ratio of copy number) lys 1:100,000 phe 1:50,000 thr 1:25,000 dap 1:6,667 The controls are then amplified and labeled together with the samples. Examining the hybridization intensities of these controls on GeneChip arrays helps to monitor the labeling process independently from the quality of the starting RNA samples. Anticipated relative signal strength follows the order of lys < phe < thr < dap. The Poly-A RNA Control Stock and Poly-A Control Dil Buffer are provided in the GeneChip TM Poly-A RNA Control Kit to prepare the appropriate serial dilutions based on Table 2.2. This is a guideline when 100, 250 or 500 ng of total RNA is used as starting material. For starting sample amounts other than those listed here, calculations are needed in order to perform the appropriate dilutions to arrive at the same proportionate final concentration of the spike-in controls in the samples. IMPORTANT: Use non-stick RNase-free microfuge tubes to prepare all of the dilutions (not included). 12 GeneChip TM HT WT User Guide

14 Table 2.2 Serial Dilution of Poly-A RNA Control Stock Total RNA Input Amount Serial Dilutions First Dilution Second Dilution Third Dilution Fourth Dilution Volume of 4 th dilution to add total RNA 100 ng 1:20 1:50 1:50 1:10 2 μl 250 ng 1:20 1:50 1:50 1:4 2 μl 500 ng 1:20 1:50 1:50 1:2 2 μl Recommendation: Avoid pipetting solutions less than 2 µl in volume to maintain precision and consistency when preparing the dilutions. For example, to prepare the Poly-A RNA dilutions for 100ng of total RNA: 1. Add 2 µl of the Poly-A Control Stock to 38 µl of Poly-A Control Dil Buffer for the First Dilution (1:20). 2. Mix thoroughly and spin down to collect the liquid at the bottom of the tube. 3. Add 2 µl of the First Dilution to 98 µl of Poly-A Control Dil Buffer to prepare the Second Dilution (1:50). 4. Mix thoroughly and spin down to collect the liquid at the bottom of the tube. 5. Add 2 µl of the Second Dilution to 98 µl of Poly-A Control Dil Buffer to prepare the Third Dilution (1:50). 6. Mix thoroughly and spin down to collect the liquid at the bottom of the tube. 7. Add 2 µl of this third dilution to 18 µl of Poly-A Control Dilution Buffer to prepare the Fourth Dilution (1:10). NOTE: The first dilution of the Poly-A RNA controls can be stored up to six weeks in a frost-free freezer at 20 C and freeze-thawed up to eight times. 8. Mix thoroughly and spin down to collect liquid at the bottom of tube. 9. Add 2 µl of this Fourth Dilution to 100 ng of Total RNA directly. The final volume of Total RNA with the diluted Poly-A controls should not exceed 5 µl. Preparation of Total RNA Plates for Processing on Automated Systems The Target Preparation protocol starts with 5 µl of material in a Bio-Rad 96-Well Hard-Shell PCR Plate. This plate can either be prepared offline (manually) or on the Beckman Coulter Biomek TM FX P Target Prep Express (TPE) System, as described below. User-prepared Plate Preparation Preparing Samples with PolyA Controls Pipet 3 µl of the total RNA sample ( ng) and 2 µl of the appropriate PolyA spike control solution into the 96-well plate. Samples should be placed into the plate in a column-wise fashion starting from the left side of the plate. For example, if preparing 24 samples, pipet the samples into the sample wells for columns 1, 2, and 3. Optional Running without PolyA Controls We highly recommend the use of PolyA controls as described above. However, if controls are not used, the total RNA sample in the Bio-Rad 96-Well Hard-Shell PCR Plate must be adjusted to a final volume of 5 µl. GeneChip TM HT WT User Guide 13

15 Chapter 3 Labware and Reagents Required This chapter provides a check list of required labware and reagents. The information is presented as follows: Equipment, Consumables, Labware and Reagents Required Labware Reagent & Labware Requirements (Part 1 to Part 6) Equipment, Consumables, Labware and Reagents Required Equipment and Labware Required Table 3.1 Beckman Coulter Biomek FX P Target Prep Instrument Product Quantity Source P/N Beckman Coulter Biomek FX Target Prep Instrument P 1 Beckman Coulter Table 3.2 Installation Kit A83103 Component Quantity Source P/N Installation Kit consists of: HT WT Reservoir Stickers 1 Set HT WT Cold Block Template 1 Alpaqua Magnetic Plate 1 Thermo Fisher Alpillo TM Plate Cushion 1 WT ATP Biomek method file (and support files) Installed by FAS Arched Auto-Sealing Lids With Wide Tabs Pkg (Pkg of 4) Table 3.3 and Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Component and Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express - Labware sufficient for 4x24 or 4x96 Rxn runs consists of: Number per Kit 96-Well Deep Well Microplate, polypropylene, 2 ml, non-sterile 8 Greiner U-bottom (round) Plate, polypropylene 8 Bio-Rad Hard Shell PCR Plate, blue shell/clear well 72 Greiner UV Star Plate, non-sterile, clear, flat bottom 8 Costar 96-Well U-bottom plate, polystyrene Well Plate, 10 ml, square well, pyramid bottom, polypropylene 4 Auto-sealing Microplate Lid with Microseal P Pad 2 Source Thermo Fisher P/N GeneChip TM HT WT User Guide

16 Table 3.4 Biomek TM Tips and Universal labware Product Packaging Total 8x Biomek Span P50 Barrier Tips (Span 8 Pipettor) (magenta) 96 ea./box- 10 box/case Biomek AP96 P250 Barrier Tips 96 ea./box - Sterile 10 box/case (AP 96 Multichannel Pipettor) (aqua) Biomek Span P250 Barrier Tips (Span 8 Pipettor) (green) Biomek Span P1000 LLS Barrier Tips (Span 8 Pipettor) (yellow) Quarter Reservoir, Divided by Width 96 ea./box - 10 box/case 96 ea./box - 5 box/case Total 16x Total 24x Total 96x Source 10 Boxes 10 Boxes 10 Boxes 10 Boxes Beckman Coulter 8 Boxes 8 Boxes 8 Boxes 8 Boxes Beckman Coulter 1 Box 1 Box 1 Box 1 Box Beckman Coulter 2 Boxes 2 Boxes 2 Boxes 2 Boxes Beckman Coulter 40 unit/case Beckman Coulter Quarter Reservoir 40 unit/case Beckman Coulter Frame for Reservoirs 1 ea. reusable reusable Beckman Coulter P/N A Reagents Required Table 3.5 HT WT Express Reagents, Miscellaneous Reagents, and Materials Product Source P/N Ambion TM WT Expression Kit for High Throughput Robotics Kit packaging options: Ambion 1 x 24 reaction Kit x 96 reaction Kit HT WT Terminal Labeling and Controls Kit* (Sufficient for 24 Rxn) Contains: Thermo Fisher GeneChip TM HT WT Terminal Labeling Kit, P/N (1 x 24 Rxn Kit) GeneChip TM Hybridization Control Kit, P/N (30 Rxn) GeneChip TM Poly-A Control Kit, P/N (100 Rxn) HT WT Terminal Labeling and Controls Kit* (Sufficient for 96 Rxn) Contains: Thermo Fisher GeneChip TM HT WT Terminal Labeling Kit, P/N (1 x 96 Rxn Kit) GeneChip TM Hybridization Control Kit, P/N (150 Rxn) GeneChip TM Poly-A Control Kit, P/N (100 Rxn) GeneChip TM Hybridization, Wash and Stain Kit (30 Rxns, for cartridges) Thermo Fisher GeneTitan TM Hybridization, Wash and Stain Kit for WT Array Plates (96 Rxns) Thermo Fisher DNAZap Ambion 9890 RNaseZap Ambion % ETOH various Isopropanol various * Individual components available for purchase separately. Labware GeneChip TM HT WT User Guide 15

17 Table 3.6 shows the types of labware used in the GeneChip HT WT Assay. For labware requirements specific to each part of the assay, see Reagent & Labware Requirements (Part 1 to Part 6). Table 3.6 Labware used on the Biomek Workstation Deck Labware Biomek AP96 P250 Barrier Tips (aqua box; pre-sterile, barrier) Supplier & Part Number Beckman Coulter P/N Labware Image Biomek Span P1000 LLS Barrier Beckman Coulter Tips P/N (yellow box; pre-sterile, barrier, conductive) Biomek Span P250 Barrier Tips (green box; pre-sterile, barrier) Beckman Coulter P/N Biomek Span P50 Pipette Tips (magenta box; pre-sterile, barrier) Beckman Coulter P/N A GeneChip TM HT WT User Guide

18 Table 3.6 Labware used on the Biomek Workstation Deck (Continued) Labware Supplier & Part Number Labware Image Bio-Rad Hard Shell 96-Well Plate (available in multiple colors) Bio-Rad P/N HSP9631 (A component of the and Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 72) Lid, Autosealing Microplates, Arched with Microseal, P Pad Bio-Rad P/N MSL-2032 (A component of the and Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 2) AbGene 96 Square Well Storage Plate (2.2 ml) AbGene P/N AB-0932 (A component of the and Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 8) 24 well mixing plate, 10 ml, square well, pyramid bottom Seahorse Bioscience P/N S30024 (A component of the Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 4) GeneChip TM HT WT User Guide 17

19 Table 3.6 Labware used on the Biomek Workstation Deck (Continued) Labware Greiner UV Star Plate Supplier & Part Number Greiner P/N (A component of the Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 8) Labware Image Greiner U-Bottom (Round), Polypropylene Plate Greiner P/N (A component of the and Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 8) Costar Polystyrene 96-well, U-bottom Plate (non-sterile) Costar P/N 3798 (A component of the Ambion TM HT WT Expression Assay Consumables for Biomek TM FX P Target Prep Express Kit P/N Kit = 16) Alpaqua Magnetic Plate Part of the HT WT Installation Kit from P/N GeneChip TM HT WT User Guide

20 Table 3.6 Labware used on the Biomek Workstation Deck (Continued) Labware Reservoir Frame Supplier & Part Number Beckman Coulter P/N Labware Image Quarter Reservoirs Quarter module, 40 ml capacity Quarter module divided by width, 19 ml capacity each receptacle Beckman Coulter P/N (40 ml) P/N (19 ml) Undivided Divided by width 40 ml capacity 19 ml capacity Reagent Cold Block, must be chilled to 4 C before use Beckman Coulter P/N A83054 A1 Metal posts on block circled in red Reagent Cold Block Template P/N The template is designed Part of the HT WT specifically for use with the Installation kit from GeneChip HT WT Reagent Kit and P/N fits precisely onto the top of the chilled cold block. Using the cold block template helps ensure the proper placement of reagent tubes onto the block for each part of the assay. See Figure 3.1 for further detail GeneChip TM HT WT User Guide 19

21 Table 3.6 Labware used on the Biomek Workstation Deck (Continued) Labware Modular Reservoir Template Stickers Modular reservoir template stickers are available to place on the front of the modular reservoir frame to, assist with the proper placement of modular reservoirs and reagents. See Figure 3.2 for further detail. Supplier & Part Number P/N Part of the HT WT Installation kit from P/N Labware Image 24-Position Tube Rack Beckman Coulter P/N (rack) P/N (insert) Tube rack with 24 inserts Adaptor, Thin PCR (installed on the Shaking Peltier) This adaptor is typically installed by a Beckman Coulter field service technician during new system installation or a system upgrade. Ensure that you have one of these adaptors on the deck prior to running this assay. Beckman Coulter P/N A GeneChip TM HT WT User Guide

22 Table 3.6 Labware used on the Biomek Workstation Deck (Continued) Labware Alpillo Plate Cushion Supplier & Part Number Part of the HT WT Installation kit from P/N Labware Image GeneTitan Hyb Tray P/N (GeneTitan trays and lids are provided with the Array Plate when ordered. The P/N provided here is for identification purposes only.) GeneTitan Stain Tray P/N (GeneTitan trays and lids are provided with the Array Plate when ordered. The P/N provided here is for identification purposes only.) GeneTitan Scan Tray P/N or (GeneTitan trays and lids are provided with the Array Plate when ordered. The P/N provided here is for identification purposes only.) GeneChip TM HT WT User Guide 21

23 Labware Templates The Ambion TM WT Expression Kit and GeneChip TM HT WT Terminal Labeling Kit provide templates to help ensure the proper placement of reagents in labware. Reagent Cold Block Template Figure 3.1 Reagent Cold Block Template Modular Reservoir Template Stickers Figure 3.2 Modular Reservoir Templates 22 GeneChip TM HT WT User Guide

24 Reagent & Labware Requirements (Part 1 to Part 6) Reagent and Labware Required for Part 1 Table 3.7 Part 1 Reagent Requirements Ambion TM WT Expression Kit for High Throughput Robotics Components needed: 1st Strand Enzyme Mix 1st Strand Buffer Mix 2nd Strand Enzyme Mix 2nd Strand Buffer Mix IVT Enzyme Mix IVT Buffer Mix Nuclease-free Water DNAZap RNaseZap * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. Description Source P/N Ambion (24 rxns)* or (96 rxns) NOTE: To use a 24 rxn Ambion TM WT Expression Kit for High Throughput Robotics for two 8 sample runs, please refer to Appendix A, Using the Ambion TM 24 Rxn Kit for Two 8-Sample Runs. Table 3.8 Part 1 Labware Requirements 8x 16x 24x 96x Description Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage Reagent Cold Block template: P/N , kept in 4 C storage 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 3 boxes 3 boxes 3 boxes 3 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A box 1 box 1 box 1 box Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032 or P/N Alpaqua Magnetic Plate: P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N GeneChip TM HT WT User Guide 23

25 Reagent and Labware Required for Part 2 Table 3.9 Part 2 Reagent Requirements Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: Nucleic Acid Binding Buffer Concentrate Nucleic Acid Binding Beads Nucleic Acid Wash Concentrate Elution Solution Nuclease-free Water Isopropanol * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. NOTE: All components and reagents, except beads, for part 2 and part 4 should be at room temperature. Allow samples to sit at room temperature for at least 10 minutes. The Bead Binding Buffer will precipitate if stored at 4 C. Ensure that the binding buffer and beads are at room temperature prior to starting the procedure. Table 3.10 Part 2 Labware Requirements 8x 16x 24x 96x Description 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 2 boxes 2 boxes 2 boxes 2 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N boxes 2 boxes 2 boxes 2 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Alpaqua Magnetic Plate: P/N Costar U-bottom 96-well, Polystyrene Plate: Costar P/N 3798 or P/N AbGene 96 Square Well Storage Plate: AbGene P/N AB-0932 or P/N Greiner UV Star Plate: Greiner P/N or P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Quarter Reservoir Divided By Width: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N GeneChip TM HT WT User Guide

26 Reagent and Labware Required for Part 3 Table 3.11 Part 3 Reagent Requirements Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: Random Primers Second-Cycle Enzyme Second Cycle Buffer RNaseH * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. Table 3.12 Part 3 Labware Requirements 8x 16x 24x 96x Description Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage 0 tips 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 0 tips Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 3 boxes 3 boxes 3 boxes 3 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N GeneChip TM HT WT User Guide 25

27 Reagent and Labware Required for Part 4 Table 3.13 Part 4 Reagent Requirements Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: Nucleic Acid Binding Buffer Concentrate Nucleic Acid Binding Beads Nucleic Acid Wash Solution Concentrate Elution Solution Nuclease-free Water DNAZap RNaseZap * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. NOTE: All components and reagents, except beads, for part 2 and part 4 should be at room temperature. Allow samples to sit at room temperature for at least 10 minutes. The Bead Binding Buffer will precipitate if stored at 4 C. Ensure that the binding buffer and beads are at room temperature prior to starting the procedure. Table 3.14 Part 4 Labware Requirements 8x 16x 24x 96x Description 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 2 boxes 2 boxes 2 boxes 2 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A boxes 2 boxes 2 boxes 2 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Alpaqua Magnetic Plate: P/N Costar U-bottom 96-well, Polystyrene Plate: Costar P/N 3798 or P/N AbGene 96 Square Well Storage Plate: AbGene P/N AB-0932 or P/N Greiner UV Star Plate: Greiner P/N or P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Quarter Reservoir Divided By Width: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N GeneChip TM HT WT User Guide

28 Reagent and Labware Required for Part 5 Table 3.15 Part 5 Reagent Requirements Per Run Description 1 GeneChip HT WT Terminal Labeling Kit: P/N (24 Rxn*) P/N (96 Rxn) Components needed: 10X cdna Fragmentation Buffer UDG, 10U/uL APE 1, 1000 U/uL 5X TdT Buffer TdT, 30 U/uL DNA Labeling Reagent, 5 mm RNase-free Water see Table 3.16 GeneChip Hybridization Control Kit: P/N (30 Rxn) or P/N (150 Rxn) see Table 3.16 GeneChip Hybridization, Wash, and Stain Kit: (30 reactions) P/N or GeneTitan Hybridization, Wash, and Stain Kit for WT Array Plates: (96 Rxn) P/N Nuclease-free Water DNAZap RNaseZap * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. Table 3.16 Part 5 Reagent Requirements Sample Format 8*, 16 and 24 Rxn 96 Rxn Array Option: Cartridge Plates Cartridge Plates Hybridization, Wash and Stain Kit 30 Rxn - P/N Hybridization Control Kit 30 Rxn - P/N * Cartridge option only for 8-sample reaction. 96 Rxn - P/N X 30 Rxn - P/N Rxn - P/N Rxn - P/N Rxn - P/N Rxn - P/N GeneChip TM HT WT User Guide 27

29 Table 3.17 Part 5 Labware Requirements - Cartridges 8x 16x 24x 96x Description Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage Reagent Cold Block template: P/N , kept in 4 C storage 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 3 boxes 3 boxes 3 boxes 3 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N Alpaqua Magnetic Plate: P/N Greiner U-Bottom 96 well Polypropylene Plate: Greiner P/N or P/N Position Tube Rack: Beckman Coulter P/N (24 Position Rack), P/N (White 10mm insert) Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N GeneChip TM HT WT User Guide

30 Table 3.18 Part 5 Labware Requirements - Plates 8x 16x 24x 96x Description N/A Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage N/A Reagent Cold Block template: P/N , kept in 4 C storage N/A 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N N/A 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) N/A 3 boxes 3 boxes 3 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N N/A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N N/A Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL or P/N N/A Alpaqua Magnetic Plate: P/N N/A Position Tube Rack: Beckman Coulter P/N (24 Position Rack), P/N (White 10mm insert) N/A Alpillo Plate Cushion: P/N N/A well mixing plate: Seahorse Bioscience P/N S30024 or P/N N/A Quarter Reservoir: Beckman Coulter P/N N/A Reservoir Frame: Beckman Coulter P/N Reagent and Labware Required for Part 6 Table 3.19 Part 6 Reagent Requirements 8x 16x 24x 96x Description GeneTitan Hybridization, Wash and Stain Kit for WT Array Plates: P/N Components needed: Array Holding Buffer Stain 1 & 3 Stain 2 Table 3.20 Part 6 Labware Requirements 8x 16x 24x 96x Description N/A 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N N/A 1 box 1 box 1 box Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N N/A Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N GeneChip TM HT WT User Guide 29

31 8x 16x 24x 96x Description N/A Alpaqua Magnetic Plate: P/N N/A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N N/A GeneTitan Hyb Tray: P/N * N/A GeneTitan Stain Tray: P/N * N/A GeneTitan Scan Tray: P/N or * N/A GeneTitan Stain/Scan Tray Lids: P/N * N/A Quarter Reservoir: Beckman Coulter P/N N/A Reservoir Frame: Beckman Coulter P/N * GeneTitan trays and lids are provided with the Array Plate when ordered. The P/N provided here is for identification purposes only. 30 GeneChip TM HT WT User Guide

32 Chapter 4 Beckman Coulter Biomek TM FX P Express Setup and Target Preparation This chapter explains how to perform WT Expression and Terminal Labeling using the Beckman Coulter Biomek TM FX P Target Prep Express (TPE) System. It also includes instructions for target preparation for processing on the GeneTitan TM Instrument. The protocol is performed on two instruments: Target preparation is performed on the Biomek FX P Target Prep Express Array processing is performed on the GeneTitan TM Instrument (Refer to the GeneTitan TM User Guide for Expression Array Plates (P/N ) for instruction on processing array plates using the GeneTitan Instrument) This chapter includes instructions for target preparation. These instructions are presented as follows: Part 1 - First and Second-Strand cdna Synthesis, IVT crna Synthesis Part 2 - crna Purification Part 3 - Second Cycle cdna Synthesis & RNase H Hydrolysis Part 4 - cdna Purification Part 5 - cdna Fragmentation, Labeling, & Hybridization Setup Part 6 - GeneTitan TM Instrument Hybridization Preparation Part 1 - First and Second-Strand cdna Synthesis, IVT crna Synthesis Approximate Duration Table 4.1 Approximate Time Required for Part 1 Part 1 - First & Second-Strand cdna Synthesis, IVT crna Synthesis 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: First strand synthesis Thermal cycler first strand method Biomek TM FX P TPE System pipetting steps: Second strand synthesis Thermal cycler second strand method Biomek TM FX P TPE System pipetting steps: IVT Thermal cycler: IVT method Part 1 Total Time 1180 min 1182 min 1185 min 1218 min ~19.7 hr 19.7 hr hr 20.3 hr GeneChip TM HT WT User Guide 31

33 Reagents and Labware Required Reagents Required Follow the guidelines below when instructed to thaw, vortex and spin buffer reagent tubes. For enzyme reagent tubes, spin only. Vortex Reagents 3 times, 1 sec each time at the maximum setting. Spin Reagent Vials 3 sec CAUTION: Carefully observe the buffer tubes after vortexing. If a precipitant is present, additional vortexing is required. Vortex until all precipitant is dissolved. Heat at 37 to 40 C if necessary. Table 4.2 Part 1 Reagent Requirements Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: 1st Strand Enzyme Mix 1st Strand Buffer Mix 2nd Strand Enzyme Mix 2nd Strand Buffer Mix IVT Enzyme Mix IVT Buffer Mix Nuclease-free Water DNAZap RNaseZap * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. 32 GeneChip TM HT WT User Guide

34 Labware Required Table 4.3 Part 1 Labware Requirements 8x 16x 24x 96x Description Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage Reagent Cold Block template: P/N , kept in 4 C storage 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 3 boxes 3 boxes 3 boxes 3 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A box 1 box 1 box 1 box Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N Alpaqua Magnetic Plate: P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N Clean the Bio-Rad 96-Well Hard-Shell PCR Plate Lids NOTE: The disposable pad under the arched lids should be cleaned before every run. Material required Ambion DNAZap Ambion RNaseZap Kimwipes Procedure 1. Rinse the pad with DI water. 2. Wipe the pad with RNaseZap. 3. Rinse the pad with DI water. 4. Wipe the pad with DNAZap. 5. Thoroughly rinse the pad with DI water. 6. Dry the pad with pressurized clean air or nitrogen. GeneChip TM HT WT User Guide 33

35 NOTE: The disposable pad under the arched lids should be replaced every 25 runs. Prepare the Biomek FX P TPE System Before beginning a sample preparation run, you must make the following checks of the Biomek FX P TPE System. Figure 4.1 Biomek TM FX P TPE System Check the Water and Waste Containers To check the system water and waste containers: 1. If necessary, fill the system water container using distilled, deionized water (ultra-pure water not required). 2. If necessary, empty the system waste bottle. 3. If necessary empty the tip waste container. 34 GeneChip TM HT WT User Guide

36 Turn on the Biomek FX P Target Prep Express To turn on the workstation: 1. Power on the workstation. 2. Ensure that all of the peripherals are powered on. Watlow temperature controllers control the Static Peltier (Pelt_1) and the Shaking Peltier (SPelt_96); no additional power supply. Thermal cycler: BIO-RAD PTC-200 with DNA Engine, or Whatman Biometra TRobot CAUTION: If using a PTC-200 thermal cycler, before turning it on confirm that the Multichannel head (pod_1) is not positioned over the thermal cycler. 3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop. You can also open Start All Programs Beckman Coulter Biomek Software. NOTE: The instructions that follow pertain to the PTC-200 thermal cycler. If using a Biometra TRobot proceed to Home All Axes. NOTE: For detailed instructions on how to use either the PTC-200 or the Biometra TRobot, please refer to the appropriate Beckman Coulter user manual for your specific thermal cycler. GeneChip TM HT WT User Guide 35

37 Close the PTC-200 Thermal Cycler Lid The PTC-200 lid may remain open upon startup. You must close the PTC-200 lid prior to homing the axes or starting a method. Close the PTC-200 lid: if it remains open after you have powered on the workstation. if the lid is up before you home the axes or before you begin a method. If not closed, the MC Pod may collide with the PTC-200 lid and damage the instrument. To close the PTC-200 lid: 1. Select Instrument Device Editor (Figure 4.2). Figure 4.2 Biomek Software - Device Editor 2. Open the Device drop-down menu and select PTC-200 Left (Figure 4.3). Figure 4.3 Device drop-down menu 3. Click Action Commands. 4. Select the following (Figure 4.4): 1. In the Actions box, select Close. 2. In the Open/Close box, select Without plate. 36 GeneChip TM HT WT User Guide

38 IMPORTANT: It is critical that you select Without plate in the Open/Close box. If a plate is present, remove it before closing the lid. 5. Click Close Lid. A Status window is displayed while the lid is being closed (Figure 4.5). 6. Click OK when the Command executed prompt is displayed (Figure 4.5). 7. Click Cancel; then click Close. Figure 4.4 Closing the PTC-200 thermal cycler lid Figure 4.5 Closing PTC-200 Lid prompts. GeneChip TM HT WT User Guide 37

39 Home All Axes This procedure will home all axes and prime the fluidics lines. To home all axes: 1. Open Instrument Home All Axes. 3. Ensure all conditions in the Warning prompt Figure 4.6 (1.) are met, then click OK. A message appears, instructing you to stop and wait during instrument homing (2.). Figure 4.6 Homing all axes 4. When: 1. The Warning prompt in Figure 4.7 (A.) is displayed, confirm that no tips are loaded in the Span- 8 Pod, and click OK. The lines for the Span-8 tips are primed and the next prompt shown in Figure 4.7 (B.) is displayed. 2. When the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK. The Biomek FX P TPE System is ready for use. Figure 4.7 Prompts displayed for priming the Span-8 Pod fluidic lines. Related Biomek FX P Target Prep Express Documentation The following user manuals are installed at the same time as the Biomek FX P software (Start All Programs Beckman Coulter Manuals). Refer to these for troubleshooting the Biomek instrument. Biomek TM Liquid Handler User s Manual, Beckman Coulter P/N Biomek TM Software User s Manual, Beckman Coulter P/N GeneChip TM HT WT User Guide

40 Breaking the Light Curtain While Running the Assay For your safety, the Biomek FX P Target Prep Express is equipped with a light curtain (Figure 4.8) that senses when an object (such as a hand or an arm) enters the space surrounding the deck. When the curtain is broken, the Biomek FX P Target Prep Express immediately halts all movement on the deck until you click OK to resume the activity that was taking place, or abort the activity. Incubation timers are not interrupted. Figure 4.8 Prompt when the light curtain is broken during the process Homing All Axes Thaw and Prepare the Samples and Reagents Thaw and Prepare the Sample Plate If the sample plate is frozen: 1. Thaw the sample plate. 5. Spin down at 1000 rpm for 30 sec. 6. To avoid cross-contamination, remove the seal carefully. GeneChip TM HT WT User Guide 39

41 Thaw and Prepare the Reagents IMPORTANT: Before loading the tubes, please mix buffer solutions by vortexing. If a precipitant is present in buffer, dissolve by placing in a heat block at 40 C, vortex, and return to heat block until dissolved. Mix enzyme solutions by gently flicking the reagent tube a few times. Briefly spin ALL tubes before inserting them into the reagent cold block. Inspect for bubbles. Wipe the bottom of the reagent cold block before placing it on the deck. To thaw and prepare the reagents: 1. Thaw the following reagents from Ambion TM WT Expression Kit on the bench top at room temperature. First Strand Buffer Second Strand Buffer IVT Buffer 7. Vortex and spin the buffers, then place on ice or in the reagent cold block. 8. Gently flick the bottom of each enzyme tube, then spin the tubes and place them on ice or in the reagent cold block with the buffers. Start the Biomek Software 1. If necessary, start the Biomek FX P Software by clicking the BioMek Software icon on the desktop. Alternately, select Start All Programs Beckman Coulter Biomek Software from the menu bar 2. Select the WT Target Prep Project. 1. Select Project Open Project on the menu bar. 2. In the dialog box that appears (Figure 4.9), select WT Target Prep and click OK. Figure 4.9 Select the WT Target Prep Project 3. Open the WT Target Prep method. 1. Click the Open button. Alternately, select File Open from the menu bar.the Open Method dialog box appears (Figure 4.10). 2. Confirm that the WT Target Prep project is selected in the Look in drop-down list. 40 GeneChip TM HT WT User Guide

42 Figure 4.10 Open Method dialog box 9. Select the WT Target Prep method and click OK. The Method View is populated with information (Figure 4.11). Figure 4.11 WT Method loaded 10. In the Method View directory tree, click WT Target Prep. 11. In the Configuration View area, under Device Configuration Option, confirm that the correct integrated thermal cycler device is selected (Bio-Rad PTC-200 or Whatman Biometra TRobot). GeneChip TM HT WT User Guide 41

43 Test Mode: The Test Mode Custom Run Option should only be used for diagnostics and not for live runs. DO NOT select Test Mode as it will disable the timers. 12. Click the Run button found under the menubar. The Run Setting Options are displayed (Figure 4.12). NOTE: After clicking Run it may take 30 to 60 seconds for method validation. Please be patient. Do not click Run again. Figure 4.12 Run Settings Options 13. Select the kit format, array format, sample format, and start step: Kit Format: Select the format (24 or 96) for the Ambion TM WT Expression Kit for High Throughput Robotics Kit you are using Array Format: Select Cartridge or Plate Sample Format: Select the sample format: 8x, 16x, 24x or 96x Start Step: Choose the starting step for the run. By default, the method starts at the beginning of Part 1 and proceeds through all subsequent steps. To choose a particular starting step, click the radio button next to the step. 14. When ready to continue, click Run. The Labware & Reagent Setup window appears (Figure 4.13). 42 GeneChip TM HT WT User Guide

44 Begin Part 1: First and Second-Strand cdna Synthesis, IVT crna Synthesis 1. After clicking Run (Step 9) the Labware & Reagent Setup window appears (Figure 4.13). IMPORTANT: At position P11 there is an Alpillo Plate Cushion under the 24 well mixing plate. Please see Table 4.4 for labware setup details. Figure 4.13 Part 1 Labware & Reagent Setup 15. Set up the reagent cold block as shown in Figure 4.13 or Figure 4.14 A or B. IMPORTANT: All reagents should already be mixed and spun down prior to loading in the reagent cold block prechilled to 4 C. 1. Place the reagent cold block template onto the cooled (4 C) reagent cold block. 2. Uncap the First-Strand, Second-Strand, and IVT reagent tubes and insert them into the prechilled cold block following the yellow color code of the template Figure Confirm that deck position Pelt_1 is set to 4 C (look at the display of the Watlow controllers below the deck), then place the cold block with the reagents on the deck at position Pelt_1. GeneChip TM HT WT User Guide 43

45 Figure 4.14 Reagent Cold Block Setup for 8, 16, 24 and 96 Reactions - Part Place the labware and Modular Reservoir on the deck as directed in the following figures and table: Figure 4.13 illustration of the deck layout Figure 4.15 location names of empty deck positions Table 4.4 table detailing the labware and Modular Reservoir placement for the deck layout Figure 4.15 Empty Deck Positions 44 GeneChip TM HT WT User Guide

46 Table 4.4 Labware and reagent locations on the deck for Part 1: First & Second-Strand cdna Synthesis, IVT crna Synthesis Position on Deck* Labware Use Reagent or Samples TL1 Biomek AP96 P50 Barrier Tips (magenta) P50-First Strand P2 Bio-Rad Hard Shell 96-Well PCR Plate Sample Sample P3 Auto-sealing Microplate Lid with Microseal P Pad Lid P4 Alpaqua Magnetic Plate Magnetic Plate P7 Bio-Rad Hard Shell 96-Well PCR Plate First Strand Dist. P8 Bio-Rad Hard Shell 96-Well PCR Plate Second Strand Dist. P9 Bio-Rad Hard Shell 96-Well PCR Plate IVT Dist. P10 Biomek AP96 P50 Barrier Tips (magenta) P50-Span8 P11 P12 Alpillo Plate Cushion with 24 well mixing plate Reservoirs in frame: Quarter module in position 1 Positions 2, 3 and 4 are empty Pour Water into reservoir 1 8 Rxn = 3 ml 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 6 ml Mixing Reagent Reservoirs Water Empty Pelt_1 Reagent Cold Block, chilled to 4 C Cold Block See Step 2 for detailed setup instructions P13 Biomek Span P250 Barrier Tips (green) P200-Span8 P14 Biomek Span P1000 LLS Barrier Tips P1000-Span8 P15 (yellow) Biomek AP96 P250 Barrier Tips (aqua) P200-Second Strand P16 Biomek AP96 P50 Barrier Tips (magenta) P50-IVT Spelt96_1 Peltier Adapter (installed on deck) * Please refer to Figure 4.15 for empty deck positions. Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200. GeneChip TM HT WT User Guide 45

47 17. When the deck is ready, click Run in (Figure 4.13). You are prompted to purge the Pod2 syringe lines. NOTE: Please verify that you have removed:. adhesive seals, if any caps from reagent tubes on the cold block lids from the tip boxes Figure 4.16 Purge Messages 18. Click Yes in the prompt. If bubbles are still present after purging, click Purge More in the next prompt that appears. If no visible bubbles in tubing or syringes, click Skip Purge. The robot proceeds without user intervention with Part 1 through the IVT crna synthesis which is held in the thermal cycler for 16 hours. When Part 1 is complete, the message shown in Figure 4.17 appears. Figure 4.17 Message at Completion of Part Follow the instructions in the message. 1. Leave the following on deck: Span-8 tips Magnetic plate Mixing plate (24-well) Modular reservoir frame and quarter reservoir with water 46 GeneChip TM HT WT User Guide

48 2. Discard the distribution plates (PCR) (for locations of distribution plates refer to Figure 4.15) and discard multi-channel tip boxes. 3. Remove and store the reagent cold block at 4 C. NOTE: This is an optional stopping point. After Part 1 is complete, the samples can be stored at 20 C. 20. To continue on to Part 2, click Yes (see page 46 for Part 2 instructions). 21. If you do not want to continue with Part 2, click No.When ready to resume ensure that clean,fresh Span-8 tip boxes are placed on the deck and click the Run button Figure 4.17 Message at Completion of Part 1 in the window that appears (Figure4.18). GeneChip TM HT WT User Guide 47

49 Part2-cRNAPurification After the IVT synthesis is complete, the crna is purified to remove enzymes, salts, and unincorporated nucleotides. At completion, Part 2 yields quantified and normalized crna. Approximate Duration Table 4.5: Approximate Time Required for Part 2 Part 2- crna Purification 8Rxns (min) 16Rxns (min) 24Rxns (min) 96Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps:crna purification Normalization Part 2 Total Time 81min 82min 83min 109min 1.35hr 1.37hr 1.38hr 1.82hr Reagents and Labware Required Reagents Required Table 4.6 Part 2 Reagent Requirements Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: Nucleic Acid Binding Buffer Concentrate Nucleic Acid Binding Beads Nucleic Acid Wash Concentrate Elution Solution Nuclease-free Water Isopropanol Ethanol,200 proof *One24rxnkitisgood for(2)8xruns, (1)16xrunor(1)24xrun. NOTE: All components and reagents, except beads, for part 2 and part 4 should be at room temperature. Allow samples to sit at room temperature for at least 10 minutes. The Bead Binding Buffer will precipitate if stored at 4 C. Ensure that the binding buffer and beads are at room temperature prior to starting the procedure. 48 GeneChip TM HT WT User Guide

50 Labware Required Table 4.7 Part 2 Labware Requirements 8x 16x 24x 96x Description 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 2 boxes 2 boxes 2 boxes 2 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N boxes 2 boxes 2 boxes 2 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Alpaqua Magnetic Plate: P/N Costar U-bottom 96-well, Polystyrene Plate: Costar P/N 3798 or P/N AbGene 96 Square Well Storage Plate: AbGene P/N AB-0932 or P/N Greiner UV Star Plate: Greiner P/N or P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Quarter Reservoir Divided By Width: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N Perform a Pre-run Check of the Biomek FX P TPE System NOTE: The pre-run check is not required if you continue on to Part 2 without stopping the method at the end of Part 1. The following actions are the same as described under Prepare the Biomek FX P TPE System. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation. CAUTION: If using the PTC-200 thermal cycler, before turning it on confirm that the Multichannel head (pod_1) is not positioned over the thermal cycler. To perform the pre-run checklist: 1. Power on the Biomek FX P Target Prep Express and all peripherals. 2. Check the water and waste containers; replenish or empty as required. 3. Launch the Biomek Software. 4. If applicable, close the PTC-200 lid. 5. Home all axes. GeneChip TM HT WT User Guide 49

51 Prepare the Samples and Reagents Thaw and Prepare the Sample Plate If the sample plate is frozen: 1. Thaw the sample plate. 2. Spin down at 1000 rpm for 30 sec. 3. To avoid cross-contamination, remove the seal carefully. Gather the crna Purification Reagents NOTE: All components and reagents should be at room temperature. Allow samples to sit at room temperature for at least 10 minutes. The Bead Binding Buffer will precipitate if cold. Ensure that the binding buffer and beads are at room temperature prior to starting the procedure. Prepare the reagents: 1. Gather the following reagents and place them on the bench top at room temperature. Binding Beads Binding Beads Buffer Wash Solution (add ethanol prior to using following the instructions on the bottle) Elution Buffer Isopropanol Nuclease-free water 2. Vortex and mix the beads thoroughly. Make sure that no beads are clumped together at the bottom of the tube. Continue the Run - Part 2: crna Purification NOTE: If you are beginning the run at Part 2, click the Run button found below the menubar and make your selections from the Run Settings Options window that appears. Begin Part 2 at Step 2 of Continue the Run - Part 2: crna Purification. 1. If prompted, place a new box of tips as indicated in the message prompt. When ready to continue click OK. 2. The Labware & Reagent Setup for Part 2 appears (Figure 4.19). IMPORTANT: Use non-stick RNase-free microfuge tubes to prepare all of the dilutions (not included). NOTE: Keep the reagent cold block in the refrigerator when not in use. 50 GeneChip TM HT WT User Guide

52 Figure 4.19 Part 2 Labware & Reagent Setup 3. Setup 24-well plate, modular reservoir, and place labware on the deck as directed in the following figures and table: Figure 4.19 illustration of the deck layout Figure 4.20 Pipette the correct reagent volumes in the 24-well plate Figure 4.21 location names of empty deck positions Table 4.8 Pipette the correct reagent volumes in the Modular Reservoir. See Table 4.8 detailing the labware and Modular Reservoir placement for the deck layout. NOTE: If the run was stopped at the end of Part 1, manually place the sample plate at P1 without a lid and ensure the you have placed clean fresh Span-8 tip boxes on the deck before resuming Part 2. GeneChip TM HT WT User Guide 51

53 Figure Well Plate for Part 2 deck setup Figure 4.21 Empty Deck Positions Table 4.8 Labware and reagent locations on the deck for Part 2: crna Purification Position Labware Use Reagent or Samples on Deck * TL1 Biomek AP96 P250 Barrier Tips (aqua) P200 - Beads P1 Bio-Rad Hard Shell 96-Well PCR Plate Sample Sample P2 Bio-Rad Hard Shell 96-Well PCR Plate Normal P4 Alpaqua Magnetic Plate Bead Magnet P5 Costar Polystyrene 96-well, U-bottom Plate Bead Binding P6 AbGene 96 Square Well Storage Plate IPA Waste P7 Costar Polystyrene 96-well, U-bottom Plate Wash Solution P8 Bio-Rad Hard Shell 96-Well PCR Plate Eluent P9 Greiner UV Star Plate OD P10 Biomek AP96 P50 Barrier Tips (magenta) P50-Span8 P11 Alpillo Plate Cushion with 24 well mixing plate Mixing See Figure 4.20 for setup instructions 52 GeneChip TM HT WT User Guide

54 Table 4.8 Labware and reagent locations on the deck for Part 2: crna Purification (Continued) Position Labware Use Reagent or on Deck * Sample P12 Reservoirs in frame: Quarter module reservoir in position 1 and 2 Quarter module reservoir divided by width in positions 3 and 4 Pipette Water into reservoir 1 8 Rxn = 6 ml 16 Rxn = 12 ml 24 Rxn = 12 ml 96 Rxn = 36 ml Pipette Wash Solution into reservoir 2 8 Rxn = 3 ml 16 Rxn = 7 ml 24 Rxn = 7 ml 96 Rxn = 24 ml Pipette Binding Buffer into the bottom half of reservoir 3 8 Rxn = ml 16 Rxn = 1.75 ml 24 Rxn = 1.75 ml 96 Rxn = 6.25 ml Pipette Elution Solution into the top half of reservoir 4 8 Rxn = 1.5 ml 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 11 ml Pipette Ethanol into the bottom half of reservoir 4 8 Rxn = 5 ml 16 Rxn = 10 ml 24 Rxn = 10 ml 96 Rxn = 20 ml Reagent Reservoirs P13 Biomek Span P250 Barrier Tips (green) P200-Span8 P14 Biomek Span P1000 LLS Barrier Tips (yellow) P1000-Span8 P15 Biomek AP96 P250 Barrier Tips (aqua) P200-Elution p16 Biomek AP96 P50 Barrier Tips (magenta) P50-OD Spelt96_1 Bio-Rad Hard Shell 96-Well PCR Plate * Please refer to Figure 4.21 for empty deck positions. Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P When the deck is ready, click Run in Figure Elution Buffer Water Wash Solution Empty Binding Buffer s Elution Solution Iso- Propanol 5. In the prompt that appears (Figure 4.22), click Yes to begin purging the Pod2 syringe lines. If bubbles are still present after purging, click Purge More in the next prompt (Figure 4.22). If no further purging is required, click Skip Purge. The run proceeds. GeneChip TM HT WT User Guide 53

55 Figure 4.22 Prompts to Purge Pod2 Syringe Lines The Biomek FX P TPE System begins the crna Purification method and proceeds through elution. After the a blank OD plate is prepared (contains diluent only), a message appears prompting you to read the plate (Figure 4.23). Figure 4.23 Prompt to read OD plate background 6. Remove the OD plate from position P9 on the deck. 7. Read background OD in a plate reader at 260 nm. 8. Export or save background OD data for later use. 9. After the plate is read, remove it from the plate reader and place it on deck position P9. Click OK in the Biomek Software prompt (Figure 4.24) to proceed with sample transfer into the OD plate. 54 GeneChip TM HT WT User Guide

56 Figure 4.24 Prompt to read OD plate background 10. Click OK after removing OD plate from P9 when prompted again to read OD plate (Figure 4.25). Figure 4.25 NOTE: The WT Quant and Norm.xls template (Figure 4.26) will open in the background waiting for OD data. 11. Read sample OD in plate reader at 260 nm. 12. Export or save sample OD data. 13. Return to the WT Quant and Norm.xls template (Figure 4.26). GeneChip TM HT WT User Guide 55

57 Figure 4.26 WT Quant and Norm.xls template 14. Click the Import Form Data button in the Quantitation and Normalization Calculations Template. The Raw Data Form worksheet is shown (Figure 4.27). Figure 4.27 Raw Data Form worksheet 15. Copy and paste background OD data saved in Step 8 above into Section 1 of the Raw Data Form (Figure 4.28). 56 GeneChip TM HT WT User Guide

58 Figure 4.28 Paste Raw Background Data 16. Copy and paste sample OD data saved in Step 12 above into Section 2 of the Raw Data Form (Figure 4.29). Figure 4.29 Paste Raw Sample Data 17. Press the Copy Adjusted Data to Template button (Figure 4.29) to copy the adjusted OD values to the template GeneChip TM HT WT User Guide 57

59 Figure Check the yield by clicking the Data Summary tab. The next step requires a minimum μg in 24 μl volume. CAUTION: Normalizing below the required minimum sample concentration at this point is not recommended and may yield undesired results. The normalization process can proceed, if desired. Samples which are below the minimum of 5.5 μg will be carried through the remainder of the procedure but will not be properly normalized. Hybridization results from improperly normalized samples may not be valid. Low OD readings may be due to bubbles in sample wells. Check plate and inspect the UV plate for bubbles. If bubbles are present, carefully remove them using a clean pipette tip. 19. In the Data Import tab, click Export Data to Biomek in the Quantitation and Normalization Calculations Template. If any of the samples do not meet the minimum concentration a warning message will appear (Figure 4.31). Click Yes in the prompt that appears (Figure 4.32). Figure 4.31 Warning Message 58 GeneChip TM HT WT User Guide

60 Figure 4.32 Quantitation and Normalization Calculations Template The next dialog box that appears (Figure 4.33) enables you to save a copy of the background-corrected data and the parameters used to determine sample normalization. Saving a backup copy is optional. 20. To save a backup copy of the data and normalization parameters: 1. Confirm the default folder or select a new folder. 2. Confirm the default file name or enter a new file name. 21. Click Save. Figure 4.33 Prompt to Save a Copy of the OD Data and Normalization Parameters The sample volumes and concentrations are normalized using elution buffer as the diluent. The Quantitation and Normalizations Template closes. GeneChip TM HT WT User Guide 59

61 22. Remove the OD plate from plate reader and discard. 23. When Part 2 is complete, the message shown in Figure 4.34 appears. Figure 4.34 Message at Completion of Part 2 TIP: This is an optional stopping point. After Part 2 is complete, the samples can be stored at 20 C. 24. Follow the instructions in the message. 1. Leave the following on deck: Span-8 tips Magnetic plate Mixing plate (24-well) Quarter modular reservoir with water Normal plate at deck position P2 2. Discard the following: Distribution plate (PCR, round-bottom, deep-well) Multi-channel tip boxes All modular reservoirs, except water 25. To continue on to Part 3, click Yes (see page 60). 26. If you do not want to continue with Part 3, click No. When ready to resume ensure that clean, fresh Span-8 tip boxes are placed on the deck and click the Run button in the window that appears (Figure 4.35). 60 GeneChip TM HT WT User Guide

62 Figure 4.35 Stopping/Resuming the Run Part 3 - Second Cycle cdna Synthesis & RNase H Hydrolysis Approximate Duration Table 4.9 Approximate Time Required for Part 3 Part 3 -Second Cycle cdna Synthesis 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: Second cycle denaturation Thermal cycler denaturation method Biomek TM FX P TPE System pipetting steps: Second cycle synthesis Thermal cycler synthesis method Biomek TM FX P TPE System pipetting steps: RNaseH hydrolysis Thermal cycler: RNaseH method Part 3 Total Time 195 min 197 min 197 min 218 min 3.25 hr 3.28 hr 3.28 hr 3.63 hr GeneChip TM HT WT User Guide 61

63 Reagents and Labware Required Table 4.10 Part 3 Reagent Requirements Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: Random Primers Second-Cycle Enzyme Second Cycle Buffer RNaseH Nuclease-free Water * One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. Thaw and Prepare the Reagents To thaw and prepare the reagents: 1. Thaw the following reagents from the Ambion TM WT Expression Kit on the bench top at room temperature. Random Primer Second Cycle Buffer 2. Vortex and spin the reagents, then place on ice. 3. Gently flick the bottom of each enzyme tube, then spin the tubes and place them on ice with the buffers. Labware Required Table 4.11 Part 3 Labware Requirements 8x 16x 24x 96x Description Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage 0 tips 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 0 tips Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 3 boxes 3 boxes 3 boxes 3 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N Perform a Pre-run Check of the Biomek FX P TPE System 62 GeneChip TM HT WT User Guide

64 NOTE: The pre-run check is not required if you continue on to Part 3 without stopping the method at the end of Part 2. The following actions are the same as described under Prepare the Biomek FX P TPE System. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation. To perform the pre-run checklist: 1. Power on the Biomek FX P Target Prep Express and all peripherals. 2. Check the water and waste containers; replenish or empty as required. 3. Launch the Biomek Software. 4. If applicable, close the PTC-200 lid. 5. Home all axes. Thaw and Prepare the Samples and Reagents Thaw and Prepare the Sample Plate If the sample plate is frozen: 1. Thaw the sample plate. 2. Spin down at 1000 rpm for 30 sec. 3. To avoid cross-contamination, remove the seal carefully. Continue the Run - Part 3: Second Cycle cdna Synthesis & RNase Hydrolysis NOTE: If you are beginning the run at Part 3, click the Run button found below the menubar and make your selections from the Run Settings Options window that appears. Begin Part 3 at Step2. 1. If prompted, place a new box of tips as indicated in the message prompt. When ready to continue click OK. 2. The Labware & Reagent Setup window appears (Figure 4.36). IMPORTANT: At position P11 there is an Alpillo Plate Cushion under the 24 well mixing plate. Please see Table 4.12 for labware setup details. GeneChip TM HT WT User Guide 63

65 Figure 4.36 Part 3 Labware & Reagent Setup 3. Set up the reagent cold block as shown in Figure 4.36 or Figure 4.37 A or B. 1. Place the reagent cold block template onto the cooled (4 C) cold block. 2. Uncap the Random Primers, Second Cycle Enzymes, Second Cycle Buffer, and RNase H reagent tubes and insert them into the cooled reagent cold block following the blue color code of the template (Figure 4.37). Place the reagent cold block on the deck at position Pelt_1. IMPORTANT: Before loading the tubes, please mix buffer solutions by vortexing. If a precipitant is present in buffer, dissolve by placing in a heat block at 40 C, vortex, and return to heat block until dissolved. Mix enzyme solutions by gently flicking the reagent tube a few times. Briefly spin ALL tubes before inserting them into the reagent cold block. Inspect for bubbles. Wipe the bottom of the reagent cold block before placing it on the deck.. 64 GeneChip TM HT WT User Guide

66 Figure 4.37 Reagent Cold Block Setup for 8, 16, 24 and 96 Reactions for Part 3 4. Place the labware and Modular Reservoir on the deck as directed in the following figures and table: Figure 4.36 illustration of the deck layout Figure 4.38 location names of empty deck positions Table 4.12 table detailing the labware and Modular Reservoir placement for the deck layout Figure 4.38 Empty Deck Positions Table 4.12 Labware and reagent locations on the deck for Part 3: Second Cycle cdna Synthesis & RNase H Hydrolysis Position Labware Use Reagent or Samples on Deck * P2 Bio-Rad Hard Shell 96-Well PCR Plate Normal Normalized crna from Part 2 P3 Auto-sealing Microplate Lid with Microseal P Pad Lid P4 Alpaqua Magnetic Plate Magnetic Plate P7 Bio-Rad Hard Shell 96-Well PCR Plate Random Primers P8 Bio-Rad Hard Shell 96-Well PCR Plate Second Cycle MM GeneChip TM HT WT User Guide 65

67 Table 4.12 Labware and reagent locations on the deck for Part 3: Second Cycle cdna Synthesis & RNase H Hydrolysis (Continued) Position on Deck* Labware Use Reagent or Samples P9 Bio-Rad Hard Shell 96-Well PCR Plate RNaseH P10 Biomek AP96 P50 Barrier Tips (magenta) P50-Span8 P11 P12 Alpillo Plate Cushion with 24 well mixing plate Reservoirs in frame: Quarter module in position 1 Positions 2, 3 and 4 are empty Pour Water into reservoir 1 8 Rxn = 3 ml 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 6 ml Mixing Reagent Reservoirs Water Pelt_1 Reagent Cold Block, chilled to 4 C Cold Block See Step 3 for detailed setup instructions P13 Biomek Span P250 Barrier Tips (green) P200-Span8 P14 Biomek Span P1000 LLS Barrier Tips (yellow) P1000-Span8 P15 Biomek AP96 P250 Barrier Tips (aqua) P50-Sample P16 Biomek AP96 P50 Barrier Tips (magenta) P50-RNaseH Spelt96_1 Peltier Adapter (installed on deck) *Please refer to Figure 4.38 for empty deck positions. Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P When the deck is ready, click Run. You are prompted to purge the Pod2 syringe lines. Figure 4.39 Purge Messages 6. If bubbles are present in the Pod2 syringe lines, click Yes to begin purging. 7. If necessary, click Purge More in the next prompt (Figure 4.22). If no further purging is required, click Skip Purge. No user intervention is required in Part 3 if you are using an integrated thermal cycler. The run proceeds without further user intervention. When Part 3 is complete, the message shown in Figure 4.40 appears. 66 GeneChip TM HT WT User Guide

68 Figure 4.40 Message at Completion of Part 3 TIP: This is an optional stopping point. After Part 3 is complete, the samples can be stored at 20 C. 8. Follow the instructions in the message. 1. Leave the following on deck: Span-8 tips Magnetic plate Mixing plate (24-well) Modular reservoir frame 2. Quarter reservoir with water 3. Discard the distribution plates (PCR) and multi-channel tip boxes 4. Remove and store the reagent cold block at 4 C. 9. To continue on to Part 4, click Yes. 10. If you do not want to continue the run, click No. When ready to resume ensure that clean, fresh Span- 8 tip boxes are placed on the deck and click the Run button in the window that appears (Figure 4.35). GeneChip TM HT WT User Guide 67

69 Figure 4.41 Stopping/Resuming the Run 68 GeneChip TM HT WT User Guide

70 Part 4 - cdna Purification Approximate Duration Table 4.13 Approximate Time Required for Part 4 Part 4 - cdna Purification 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: cdna purification Normalization Reagents and Labware Required Reagents Required Table 4.14 Part 4 Reagent Requirements Part 4 Total Time 84 min 84 min 84 min 108 min 1.40 hr 1.40 hr 1.40 hr 1.80 hr Per Run Description 1 Ambion TM WT Expression Kit For High Throughput Robotics: Ambion P/N , 24 Rxn* or P/N , 96 Rxn Components needed: Nucleic Acid Binding Buffer Concentrate Nucleic Acid Binding Beads Nucleic Acid Wash Concentrate Elution Solution Nuclease-free Water DNAZap RNaseZap *One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. NOTE: All components and reagents, except beads, for part 2 and part 4 should be at room temperature. Allow samples to sit at room temperature for at least 10 minutes. The Bead Binding Buffer will precipitate if stored at 4 C. Ensure that the binding buffer and beads are at room temperature prior to starting the procedure. Prepare the Reagents To prepare the reagents: 1. Thaw the following reagents from the Ambion TM WT Expression Kit on the bench top at room temperature. Binding Beads Binding Beads Buffer Wash Solution (add ethanol prior to use) Elution Buffer: ethanol (200 proof) water Vortex and spin the reagents, then place on ice. GeneChip TM HT WT User Guide 69

71 Labware Required Table 4.15 Part 4 Labware Requirements 8x 16x 24x 96x Description 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 2 boxes 2 boxes 2 boxes 2 boxes Biomek Span P50 Pipette Tips (magenta): Beckman Coulter P/N A boxes 2 boxes 2 boxes 2 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Alpaqua Magnetic Plate: P/N Costar U-bottom 96-well, Polystyrene Plate: Costar P/N 3798 or P/N AbGene 96 Square Well Storage Plate: AbGene P/N AB-0932 or P/N Greiner UV Star Plate: Greiner P/N or P/N Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Quarter Reservoir Divided By Width: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N Perform a Pre-run Check of the Biomek FX P TPE System NOTE: The pre-run check is not required if you continue on to Part 4 without stopping the method at the end of Part 3. The following actions are the same as described under Prepare the Biomek FX P TPE System. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation. To perform the pre-run checklist: 1. Power on the Biomek FX P Target Prep Express and all peripherals. 2. Check the water and waste containers; replenish or empty as required. 3. Launch the Biomek Software. 4. If applicable, close the PTC-200 lid. 5. Home all axes. 70 GeneChip TM HT WT User Guide

72 Thaw and Prepare the Samples and Reagents Thaw and Prepare the Sample Plate If the sample plate is frozen: 1. Thaw the sample plate. 2. Spin down at 1000 rpm for 30 sec. 3. To avoid cross-contamination, remove the seal carefully. Continue the Run - Part 4: cdna Purification NOTE: If you are beginning the run at Part 4, click the Run button found below the menubar and make your selections from the Run Settings Options window that appears. Begin Part 4 at Step If prompted, place a new box of tips as indicated in the message prompt. When ready to continue click OK. 2. The Labware & Reagent Setup window appears (Figure 4.42). IMPORTANT: At position P11 there is an Alpillo Plate Cushion under the 24 well mixing plate. Please see Table 4.16 for labware setup details. GeneChip TM HT WT User Guide 71

73 Figure 4.42 Part 4 Labware & Reagent Setup 3. Setup 24-well plate, modular reservoir, and place labware on the deck as directed in the following figures and table: Figure 4.42 illustration of the deck layout Figure 4.43 Pipette the correct reagent volumes in the 24-well plate Figure 4.44 location names of empty deck positions Table 4.16 Pipette the correct reagent volumes in the Modular Reservoir. See Table 4.16 detailing the labware and Modular Reservoir placement for the deck layout. 72 GeneChip TM HT WT User Guide

74 Figure Well Plate for Part 4 deck setup Figure 4.44 Empty Deck Positions Table 4.16 Labware and reagent locations on the deck for Part 4: cdna Purification Position Labware Use Reagent or Samples on Deck * TL1 Biomek AP96 P250 Barrier Tips (aqua) P200-Beads P1 Bio-Rad Hard Shell 96-Well PCR Plate Hydrolyzed Sample Hydrolyzed Sample from Step 3 P2 Bio-Rad Hard Shell 96-Well PCR Plate Normal P4 Alpaqua Magnetic Plate Bead Magnet P5 Costar Polystyrene 96-well, U-bottom Plate Bead Binding P6 AbGene 96 Square Well Storage Plate EtOH-Waste P7 Costar Polystyrene 96-well, U-bottom Plate Wash Solution P8 Bio-Rad Hard Shell 96-Well PCR Plate Eluent P9 Greiner UV Star Plate OD P10 Biomek AP96 P50 Barrier Tips (magenta) P-50-Span8 P11 Alpillo Plate Cushion with 24 well mixing plate Mixing See Figure 4.43 for setup GeneChip TM HT WT User Guide 73

75 Position Labware Use Reagent or Samples on Deck * P12 Reservoirs in frame: Reagent Reservoirs Quarter module reservoir in position 1 and 2 Quarter module reservoir divided by width in positions 3 and 4 Pipette Water into reservoir 1 8 Rxn = 6 ml 16 Rxn = 12 ml 24 Rxn = 12 ml 96 Rxn = 36 ml Pipette Wash Solution into reservoir 2 8 Rxn = 3 ml 16 Rxn = 7 ml 24 Rxn = 7 ml 96 Rxn = 24 ml Pipette Binding Buffer into the bottom half of reservoir 3 8 Rxn = ml 16 Rxn = 1.75 ml 24 Rxn = 1.75 ml 96 Rxn = 6.25 ml Pipette Elution Solution into the top half of reservoir 4 8 Rxn = 1.5 ml 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 11 ml Pipette Isopropanol into the bottom half of reservoir 4 8 Rxn = 5 ml 16 Rxn = 10 ml 24 Rxn = 10 ml 96 Rxn = 20 ml P13 Biomek Span P250 Barrier Tips (green) P200-Span8 P14 Biomek Span P1000 LLS Barrier Tips (yellow) P1000-Span8 P15 Biomek AP96 P250 Barrier Tips (aqua) P200-Elution p16 Biomek AP96 P50 Barrier Tips (magenta) P50-OD Water Spelt96_1 Bio-Rad Hard Shell 96-Well PCR Plate Elution Buffer *Please refer to Figure 4.44 for empty deck positions. Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P When ready, click Run in Figure A message to purge the Pod2 syringe lines appears (Figure 4.45). Wash Solution Empty Binding Buffer Elution Solution Iso- Propanol 74 GeneChip TM HT WT User Guide

76 Figure 4.45 Prompts to Purge Pod2 Syringe Lines 5. If bubbles are present in the Pod2 syringe lines, click Yes to begin purging. If necessary, click Purge More in the next prompt (Figure 4.45). If no further purging is required, click Skip Purge. The run proceeds. At this point the Biomek FX P TPE System begins the cdna Purification method without user intervention. After the a blank OD plate is prepared (contains diluent only), a message appears prompting you to read the plate (Figure 4.46). Figure 4.46 Prompt to read OD plate background 6. Remove the OD plate from position P9 on the deck. 7. Read background OD in a plate reader at 260 nm. 8. Export or save background OD data for later use. 9. After the plate is read, remove it from the plate reader and place it on deck position P9. Click OK in the Biomek Software prompt (Figure 4.47) to proceed with sample transfer into the OD plate. GeneChip TM HT WT User Guide 75

77 Figure 4.47 Prompt to read OD plate background 10. Click OK after removing OD plate from P9 when prompted again to read OD plate (Figure 4.48) Figure 4.48 NOTE: The WT Quant and Norm.xls template (Figure 4.49) will open in the background waiting for OD data. 11. Read sample OD in plate reader at 260 nm. 12. Export or save sample OD data. 13. Return to the WT Quant and Norm.xls template (Figure 4.49). 76 GeneChip TM HT WT User Guide

78 Figure 4.49 WT Quant and Norm.xls template 14. Click the Import Form Data button in the Quantitation and Normalization Calculations Template. The Raw Data Form worksheet is shown (Figure 4.50). Figure 4.50 Raw Data Form worksheet GeneChip TM HT WT User Guide 77

79 15. Copy and paste background OD data saved in Step 8 above into Section 1 of the Raw Data Form (Figure 4.51). Figure 4.51 Paste Raw Background Data 16. Copy and paste sample OD data saved in Step 12 above into Section 2 of the Raw Data Form (Figure 4.52). Figure 4.52 Paste Raw Sample Data 17. Press the Copy Adjusted Data to Template button (Figure 4.52) to copy the adjusted OD values to the template. 78 GeneChip TM HT WT User Guide

80 Figure Check the yield by clicking the Data Summary tab. The next step requires a minimum 5.5 μg in 31.2 μl volume. CAUTION: Normalizing below the required minimum sample concentration at this point is not recommended and may yield undesired results. The normalization process can proceed, if desired. Samples which are outside the range of normalization (20 to 200 μg) will be carried through the remainder of the procedure but will not be properly normalized. Hybridization results from improperly normalized samples may not be valid. Low OD readings may be due to bubbles in sample wells. Check plate and inspect the UV plate for bubbles. If bubbles are present, carefully remove them using a clean pipette tip. 19. In the Data Import tab, click Export Data to Biomek in the Quantitation and Normalization Calculations Template. If any of the samples do not meet the minimum concentration a warning message will appear (Figure 4.54). Click Yes in the prompt that appears (Figure 4.55). Figure 4.54 Warning Message GeneChip TM HT WT User Guide 79

81 Figure 4.55 Quantitation and Normalization Calculations Template The next dialog box that appears (Figure 4.56) enables you to save a copy of the background-corrected data and the parameters used to determine sample normalization. Saving a backup copy is optional. 20. To save a backup copy of the data and normalization parameters: 1. Confirm the default folder or select a new folder. 2. Confirm the default file name or enter a new file name. 21. Click Save. Figure 4.56 Prompt to Save a Copy of the OD Data and Normalization Parameters The sample volumes and concentrations are normalized using elution buffer as the diluent. The Quantitation and Normalizations Template closes. 80 GeneChip TM HT WT User Guide

82 22. Remove the OD plate from plate reader and discard. When Part 4 is complete, the message shown in Figure 4.57 appears. Figure 4.57 Message at Completion of Part 4 TIP: This is an optional stopping point. After Part 4 is complete, the samples can be stored at 20 C. 23. Follow the instructions in the message. 1. Discard used labware and reagents. 2. Discard used multi-channel tips. 3. Leave Normal plate at P2 4. Leave Span8 tips in current locations. 24. To continue on to Part 5, click Yes. 25. If you do not want to continue to run, click No. When ready to resume ensure that clean, fresh Span-8 tip boxes are placed on the deck and click the Run button in the window that appears (Figure 4.58). GeneChip TM HT WT User Guide 81

83 Figure 4.58 Stopping/Resuming the Run 82 GeneChip TM HT WT User Guide

84 Part 5 - cdna Fragmentation, Labeling, & Hybridization Setup Approximate Duration Table 4.17 Approximate Time Required for Part 5 Part 5 - cdna Fragmentation, Labeling, and Hybridization Setup 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup Biomek TM FX P TPE System pipetting steps: cdna fragmentation Thermal cycler fragmentation method Biomek TM FX P TPE System pipetting steps: Labeling Thermal cycler labeling method Biomek TM FX P TPE System pipetting steps: Hybridization setup GeneChip TM Cartridge Array Hybridization setup Or or or or or GeneChip TM Array Plate Hybridization setup N/A Part 5 Total Time - Cartridge Array 164 min 167 min 167 min 185 min 2.73 hr 2.78 hr 2.78 hr 3.08 hr Part 5 Total Time - Array Plate N/A 168 min 168 min 186 min 2.80 hr 2.80 hr 3.10 hr Reagents and Labware Required Table 4.18 Part 5 Reagent Requirements Per Run See Table 4.19 See Table 4.19 Description 1 GeneChip HT WT Terminal Labeling Kit: P/N (24 Rxn)* P/N (96 Rxn) Components needed: 10X cdna Fragmentation Buffer UDG, 10U/uL APE 1, 1,000 U/uL 5X TdT Buffer TdT, 30 U/uL DNA Labeling Reagent, 5 mm GeneChip Hybridization Control Kit: P/N (30 Rxn) or P/N (150 Rxn) GeneChip Hybridization, Wash, and Stain Kit: (30 reactions) P/N or GeneTitan Hybridization, Wash, and Stain Kit for WT Array Plates: (96 Rxn) P/N Nuclease-free Water DNAZap RNaseZap *One 24 rxn kit is good for (2) 8x runs, (1) 16x run or (1) 24x run. GeneChip TM HT WT User Guide 83

85 Table 4.19 Sample Format 8*, 16 and 24 Rxn 96 Rxn Array Option: Cartridge Plates Cartridge Plates Hybridization, Wash and 30 Rxn - P/N Rxn - P/N Stain Kit Hybridization Control 30 Rxn - P/N Rxn - P/N Kit *Cartridge option only for 8-sample reaction. Thaw and Prepare the Reagents To thaw and prepare the reagents: 4 X 30 Rxn - P/N Rxn - P/N Rxn - P/N Rxn - P/N Thaw the following reagents from the GeneChip TM HT WT Terminal Labeling Kit on the bench top at room temperature. Fragmentation Buffer DNA Labeling Reagent TdT Buffer 2. Thaw the following reagents from the GeneChip TM Hybridization Control Kit on the bench top at room temperature. Oligo B2 20x Hyb Controls 3. (Cartridge Arrays) Remove the following reagents from the GeneChip Hybridization, Wash and Stain Kit (P/N ). 2X Hyb Mix Water DMSO (Thaw on the bench top at room temperature) 4. (Array Plates) Remove the following components from the GeneTitan Hybridization, Wash, and Stain Kit for WT Array Plates (P/N ) 5X WT Hyb Add 1 15X WT Hyb Add 4 2.5X WT Hyb Add 6 5. Vortex and spin the reagents, then place on ice. Labware Required Table 4.20 Part 5 Labware Requirements - Cartridge 8x 16x 24x 96x Description Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage Reagent Cold Block template: P/N , kept in 4 C storage 1 box 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N 1 box 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P200.) 3 boxes 3 boxes 3 boxes 3 boxes Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N GeneChip TM HT WT User Guide

86 Table 4.20 Part 5 Labware Requirements - Cartridge (Continued) 8x 16x 24x 96x Description Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N Alpaqua Magnetic Plate: P/N Greiner U-Bottom 96 well Polypropylene Plate: Greiner P/N or P/N Position Tube Rack: Beckman Coulter P/N (24 Position Rack), P/N (White 10mm insert) Alpillo Plate Cushion: P/N well mixing plate: Seahorse Bioscience P/N S30024 or P/N Quarter Reservoir: Beckman Coulter P/N Reservoir Frame: Beckman Coulter P/N Table 4.21 Part 5 Labware Requirements Plates 8x 16x 24x 96x Description N/A Reagent Cold Block: Beckman Coulter P/N A83054, kept in 4 C storage N/A Reagent Cold Block template: P/N , kept in 4 C storage N/A 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N N/A 1 box 1 box 1 box Biomek Span P250 Barrier Tips (green): Beckman Coulter P/N (Note: The P250 Barrier Pipette Tips are loaded where the user interface shows N/A 3 boxes 3 boxes 3 boxes P200.) Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N N/A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N N/A Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032, or P/N N/A Alpaqua Magnetic Plate: P/N N/A Position Tube Rack: Beckman Coulter P/N (24 Position Rack), P/N mm insert) (White N/A Alpillo Plate Cushion: P/N N/A well mixing plate: Seahorse Bioscience P/N S30024 or P/N N/A Quarter Reservoir: Beckman Coulter P/N N/A Reservoir Frame: Beckman Coulter P/N GeneChip TM HT WT User Guide 85

87 Perform a Pre-run Check of the Biomek FXP TPE System NOTE: The pre-run check is not required if you continue on to Part 5 without stopping the method at the end of Part 4. The following actions are the same as described under Prepare the Biomek FX P TPE System. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation. To perform the pre-run checklist: 1. Power on the Biomek FX P Target Prep Express and all peripherals. 2. Check the water and waste containers; replenish or empty as required. 3. Launch the Biomek Software. 4. If applicable, close the PTC-200 lid. 5. Home all axes. Thaw and Prepare the Samples and Reagents Thaw and Prepare the Sample Plate If the sample plate is frozen: 1. Thaw the sample plate. 2. Spin down at 1000 rpm for 30 sec. 3. To avoid cross-contamination, remove the seal carefully. Continue the Run - Part 5: cdna Fragmentation, Labeling & Hybridization Setup NOTE: If you are beginning the run at Part 5, click the Run button found below the menubar and make your selections from the Run Settings Options window that appears. Begin Part 5 at Step 2 under Continue the Run - Part 5: cdna Fragmentation, Labeling & Hybridization Setup.. 1. If prompted, place a new box of tips as indicated in the message prompt. When ready to continue click OK. 2. If running cartridge arrays, please go to Part 5 Deck and Reagent Setup - Cartridge Arrays. If running array plates, please go to Part 5 Deck and Reagent Setup - Array Plates. Part 5 Deck and Reagent Setup - Cartridge Arrays 1. The Labware & Reagent Setup window for your run appears (Figure 4.59). IMPORTANT: At position P11 there is an Alpillo Plate Cushion under the 24 well mixing plate. Please see Table 4.22 for labware setup details. 86 GeneChip TM HT WT User Guide

88 Figure 4.59 Part 5 Labware & Reagent Setup for Cartridge Arrays 2. Setup modular reservoir, and place labware on the deck as directed in the following figures and table: Figure 4.59 illustration of the deck layout for cartridge arrays Figure 4.60 location names of empty deck positions Table 4.22 Pipette the correct reagent volumes in the Modular Reservoir. See Table 4.22 detailing the labware and Modular Reservoir placement for the deck layout. GeneChip TM HT WT User Guide 87

89 Figure 4.60 Empty Deck Positions Table 4.22 Labware and reagent locations on the deck for Part 5: cdna Fragmentation, Labeling, & Hybridization Setup - Cartridge Position on Labware Use Reagent or Samples Deck * P1 Biomek AP96 P250 Barrier Tips (aqua) P200-Frag P2 Bio-Rad Hard Shell 96-Well PCR Plate Normal Normalized cdna sample from Part 4 P3 Auto-sealing Microplate Lid with Lid P4 Microseal Alpaqua Magnetic P Pad Plate Magnetic Plate P5 Greiner U-Bottom, Polypropylene Plate Hyb-Ready Target P7 Bio-Rad Hard Shell 96-Well PCR Plate Frag Dist. P8 Bio-Rad Hard Shell 96-Well PCR Plate Label Dist. P9 Greiner U-Bottom, Polypropylene Plate Hyb Cocktail Dist. P10 24-Position Tube Rack Hyb Reagents Setup instructions are provided in Step 3 P11 Alpillo Plate Cushion with 24 well Mixing P12 mixing Reservoirs plate in frame: Reagent Reservoirs Quarter module reservoir in position 1 and 2 2X Hyb Empty Water Positions 3 and 4 are empty Mix Pipette Water into reservoir 1 8 Rxn = 3 ml 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 5 ml Pipette Hyb Buffer into reservoir 2 8 Rxn = 3 ml 16 Rxn = 7 ml 24 Rxn = 7 ml 96 Rxn = 24 ml Pelt_1 Reagent Cold Block Cold Block Setup instructions are provided in Step 4 88 GeneChip TM HT WT User Guide

90 Table 4.22 Labware and reagent locations on the deck for Part 5: cdna Fragmentation, Labeling, & Hybridization Setup Cartridge Position on Deck* Labware Use Reagent or Samples P13 Biomek Span P250 Barrier Tips (green) P200-Span8 P14 Biomek Span P1000 LLS Barrier Tips (yellow) P1000-Span8 P15 Biomek AP96 P250 Barrier Tips (aqua) P200-Label P16 Biomek AP96 P250 Barrier Tips (aqua) P200-Hyb Spelt96_1 Peltier Adapter (installed on deck) * Please refer to Figure 4.60 for empty deck positions. Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P Set up the 24-position tube rack as shown in Figure 4.61, depending on your sample format. Figure 4.61 Tube Rack Setup - Cartridge Arrays 4. Set up the reagent cold block as shown in Figure 4.62 A or B, depending on your sample format. 1. Place the reagent cold block template onto the cooled (4 C) cold block. 2. Uncap the Fragmentation and DNA Labeling Reagent tubes and insert them into the cooled reagent cold block following the green and white color code of the template (Figure 4.62). 3. Uncap the thawed Oligo B2 and 20X Hybridization Control tubes and insert them into the cooled reagent cold block following the white color code of the template (Figure 4.62). 4. Confirm that the Static Peltier is set to 4 C, then place the cold block on deck position Pelt_1. IMPORTANT: Before loading the tubes, please mix buffer solutions by vortexing. If a precipitant is present in buffer, dissolve by placing in a heat block at 40 C, vortex, and return to heat block until dissolved. Mix enzyme solutions by gently flicking the reagent tube a few times. Briefly spin ALL tubes before inserting them into the reagent cold block. Inspect for bubbles. Wipe the bottom of the reagent cold block before placing it on the deck. CAUTION: DMSO freezes at 4 C. Please make sure it is completely thawed before placing it in the tube rack. GeneChip TM HT WT User Guide 89

91 Figure 4.62 Reagent Cold Block Setup for 8, 16, 24 and 96 Reactions for Part 5 Cartridge Arrays 5. Go to Step 1 to continue. Part 5 Deck and Reagent Setup - Array Plates 1. The Labware & Reagent Setup window for your run appears (Figure 4.63). IMPORTANT: At position P11 there is an Alpillo Plate Cushion under the 24 well mixing plate. Please see Table 4.23 for labware setup details. Figure 4.63 Part 5 Labware & Reagent Setup for Array Plates 90 GeneChip TM HT WT User Guide

92 2. Setup modular reservoir, and place labware on the deck as directed in the following figures and table: Figure 4.63 illustration of the deck layout for array plates Figure 4.64 location names of empty deck positions Table 4.23 Pipette the correct reagent volumes in the Modular Reservoir. See Table 4.23 detailing the labware and Modular Reservoir placement for the deck layout. Figure 4.64 Empty Deck Positions GeneChip TM HT WT User Guide 91

93 Table 4.23 Labware and reagent locations on the deck for Part 5: cdna Fragmentation, Labeling, & Hybridization Setup Plates Position on Labware Use Reagent or Samples Deck * P1 Biomek AP96 P250 Barrier Tips (aqua) P200-Frag P2 Bio-Rad Hard Shell 96-Well PCR Plate Normal Normalized cdna sample from Part 4 P3 Auto-sealing Microplate Lid with Lid P4 Microseal Alpaqua Magnetic P Pad Plate Magnetic Plate P5 Greiner U-Bottom, Polypropylene Plate Hyb-Ready Target P7 Bio-Rad Hard Shell 96-Well PCR Plate Frag Dist. P8 Bio-Rad Hard Shell 96-Well PCR Plate Label Dist. P9 Greiner U-Bottom, Polypropylene Plate Hyb Cocktail Dist. P10 24-Position Tube Rack Hyb Reagents Setup instructions are provided in Step 3 P11 Alpillo Plate Cushion with 24 well Mixing P12 mixing Reservoirs plate in frame: Reagent Quarter module reservoir in Reservoirs position 1 Empty Water Positions 2, 3 and 4 are empty Pipette Water into reservoir 1 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 3 ml Pelt_1 Reagent Cold Block Cold Block Setup instructions are provided in Step 4 P13 Biomek Span P250 Barrier Tips (green) P200-Span8 P14 Biomek Span P1000 LLS Barrier Tips (yellow) P1000-Span8 P15 Biomek AP96 P250 Barrier Tips (aqua) P200-Label P16 Biomek AP96 P250 Barrier Tips (aqua) P200-Hyb Spelt96_1 Peltier Adapter (installed on deck) * Please refer to Figure 4.64 for empty deck positions. Note: The P250 Barrier Pipette Tips are loaded where the user interface shows P GeneChip TM HT WT User Guide

94 3. Set up the tube rack as shown in Figure 4.65, depending on your sample format. Figure 4.65 Tube Rack Setup - Array Plates 4. Set up the reagent cold block as shown in Figure 4.66 A or B, depending on your sample format. 1. Place the reagent cold block template onto the cooled (4 C) cold block. 2. Uncap the Fragmentation and DNA Labeling Reagent tubes and insert them into the cooled reagent cold block following the green and white color code of the template (Figure 4.66). 3. Uncap the thawed Oligo B2 and 20X Hybridization Control tubes and insert them into the cooled reagent cold block following the white color code of the template (Figure 4.66). 4. Confirm that the Static Peltier is set to 4 C, then place the cold block on deck position Pelt_1. IMPORTANT: Before loading the tubes, please mix buffer solutions by vortexing. If a precipitant is present in buffer, dissolve by placing in a heat block at 40 C, vortex, and return to heat block until dissolved. Mix enzyme solutions by gently flicking the reagent tube a few times. Briefly spin ALL tubes before inserting them into the reagent cold block. Inspect for bubbles. Wipe the bottom of the reagent cold block before placing it on the deck.. GeneChip TM HT WT User Guide 93

95 Figure 4.66 Reagent Cold Block Setup for 16, 24 and 96 Reactions for Part 5 Array Plates 5. Go to Step 1 to continue. Continuing the Run After Deck and Reagent Setup 1. When the deck is ready, click OK in the deck setup window (Figure cartridge arrays or Figure array plates). A message to purge the Pod2 syringe lines appears (Figure 4.67). Figure 4.67 Prompts to Purge Pod2 Syringe Lines 2. If bubbles are present in the Pod2 syringe lines, click Yes to begin purging. If necessary, click Purge More in the next prompt (Figure 4.45). If no further purging is required, click Skip Purge. The run proceeds without user intervention. When Labeling is complete, the message shown in Figure 4.40 appears. 94 GeneChip TM HT WT User Guide

96 Figure 4.68 Message at Completion of Labeling 3. To continue on to Hybridization Setup, leave all labware on deck and click Yes. 4. If you are stopping the run, follow the instructions in the message and click No. Store hybridization ready target plate and left over labeled cdna plate at 20 C for future use. 5. When ready to resume ensure that the thermal cycler lid is closed; ensure that clean, fresh Span-8 tip boxes are placed on the deck and click the Run button., then click Run in the window that appears (Figure 4.69). GeneChip TM HT WT User Guide 95

97 Figure 4.69 Stopping/Resuming the Run The run proceeds without user intervention. If you specified the Cartridge Array sample format, the High Throughput Ambion WT Expression Assay is complete when Hybridization Setup is finished. At the completion of the method, the message in Figure 4.70 appears. Figure 4.70 Message at Completion of Part 5 (Cartridge Array Sample Format) For instructions on hybridization, washing and staining, and scanning arrays, please refer to the GeneChip Expression Wash, Stain and Scan User Manual for Cartridge Arrays (P/N ). 96 GeneChip TM HT WT User Guide

98 GeneChip Array Plates If you specified the Array Plate sample format, the method includes Part 6. When Hybridization Setup is complete, the message shown in Figure 4.71 appears. Figure 4.71 Message at Completion of Part 5 1. To continue on to Part 6, follow the instructions in the message and click Yes. 2. If you are stopping the run, follow the instructions in the message and click No. When ready to resume ensure that clean, fresh Span-8 tip boxes are placed on the deck and click the Run button., then click Run., in the window that appears (Figure 4.58). GeneChip TM HT WT User Guide 97

99 Figure 4.72 Stopping/Resuming the Run Part 6 - GeneTitan TM Instrument Hybridization Preparation At completion, Part 6 yields denatured cdna in a Hybridization Plate Tray, ready for further processing on the GeneTitan MC Instrument. The GeneTitan Stain Trays (Stain 1 to Stain 3) and Scan Tray are also prepared. Please refer to the GeneTitan TM Multi-Channel Instrument User s Guide (P/N ) or the GeneTitan TM Instrument User s Guide ( ) for instructions on the proper handling of plates processed using the GeneTitan family of instruments. Approximate Duration Table 4.24 Approximate Time Required for Part 6 Part 6 - GeneTitan Hyb Prep 8 Rxns (min) 16 Rxns (min) 24 Rxns (min) 96 Rxns (min) Deck setup N/A Biomek TM FX P TPE System pipetting steps N/A Thermal cycler: denaturation method N/A Part 6 Total Time N/A 32 min 52 min 82 min 1.37 hr 98 GeneChip TM HT WT User Guide

100 Reagents and Labware Required Reagents Required Table 4.25 Part 6 Reagent Requirements 8x 16x 24x 96x Description N/A Hybridization-ready Target Plate (from Part 5) N/A GeneTitan Hybridization, Wash and Stain Kit for WT Array Plates: P/N Components needed: Array Holding Buffer Stain 1 & 3 Stain 2 Labware Required Table 4.26 Part 6 Labware Requirements 8x 16x 24x 96x Description N/A 1 box 1 box 1 box Biomek Span P1000 LLS Barrier Tips (yellow): Beckman Coulter P/N N/A 1 box 1 box 1 box Biomek AP96 P250 Barrier Tips (aqua): Beckman Coulter P/N N/A Auto-sealing Microplate Lid with Microseal P Pad: Bio-Rad P/N MSL-2032 or P/N N/A Alpaqua Magnetic Plate: P/N N/A Bio-Rad Hard Shell 96-Well PCR Plate: Bio-Rad P/N HSP9631 or P/N N/A GeneTitan Hyb Tray: P/N * Table 4.26 Part 6 Labware Requirements (Continued) 8x 16x 24x 96x Description N/A GeneTitan Stain Tray: P/N * N/A GeneTitan Scan Tray: P/N or * N/A GeneTitan Stain/Scan Tray Lids, P/N * N/A Quarter Reservoir: Beckman Coulter P/N N/A Reservoir Frame: Beckman Coulter P/N * GeneTitan trays and lids are provided with the Array Plate when ordered. The P/N provided here is for identification purposes only. GeneChip TM HT WT User Guide 99

101 Perform a Pre-run Check of the Biomek FX P TPE System NOTE: The pre-run check is not required if you continue on to Part 6 without stopping the method at the end of Part 5. The following actions are the same as described under Prepare the Biomek FX P TPE System. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation. To perform the pre-run checklist: 1. Power on the Biomek FX P Target Prep Express and all peripherals. 2. Check the water and waste containers; replenish or empty as required. 3. Launch the Biomek Software. 4. If applicable, close the PTC-200 lid. 5. Home all axes. Labeling GeneTitan Reagent Trays When preparing the reagent trays to be loaded onto the GeneTitan Instrument, you will need to mark the front of each tray in a way that identifies its contents. IMPORTANT: It is critical that your write on the front edge of Hyb and Stain Trays only. The front edge of the tray is the short side with the lettering A through H. Do NOT write on any other side, as this can interfere with sensors inside the of GeneTitan Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan Instrument, you can also mark the notched corner of the trays and lids. Figure 4.73 Labeling GeneTitan Hyb Trays and Stain Trays Thaw and Prepare the Samples and Reagents Thaw and Prepare the Sample Plate If the sample plate is frozen: 1. Thaw the sample plate. 2. Spin down at 1000 rpm for 30 sec. 3. To avoid cross-contamination, remove the seal carefully. 100 GeneChip TM HT WT User Guide

102 Anti-Static Procedure for GeneTitan Instrument Trays and Covers Use the following technique to destatic GeneTitan Instrument Stain Tray trays and lids. IMPORTANT: Except for the HT array tray and the hybridization tray, you must deionize all GeneTitan stain trays, stain tray covers and scan tray cover using an anti-static gun. You must do this before you fill the trays with reagents and before you place the covers on the trays. Deionization removes the static electricity. The presence of static electricity on the underside of the cover can cause the gripper to lift the tray along with the tray cover and can result in an aborted run. See Figure 4.74, Figure 4.75 and Figure Deionize the inner surface of each tray and lid: The surface of the tray with the wells that will hold reagents. The surface of the lid that will cover the reagents. CAUTION: Do not deionize the scan tray or hybridization tray. Figure 4.74 Scan Tray with Cover. Deionize only the cover. GeneChip TM HT WT User Guide 101

103 Figure 4.75 Stain Tray with Cover. Deionize the cover and the tray. Testing the Anti-Static Gun Verify that the anti-static gun (PN , Figure 4.76.) is in working condition. You can use the protective cap on the gun to determine if the anti-static gun is releasing ions. The procedure is as follows: Keep the cap on the gun and press the trigger and release it. The cap should glow with each trigger operation since the charged ions are being discharged. If the cap does not glow, the gun may be unusable and you should replaced it. Each anti-static gun is capable of 50,000 trigger operations which is sufficient for approximately runs on the GeneTitan instrument. IMPORTANT: Make sure you remove the cap from the gun when you deionize a tray or cover. Deionization Procedure The following process provides guidance on how to use the anti-static gun on the stain and scan tray covers only. See Figure WARNING: The deionization steps 4 and 5 will damage the HT arrays on the plate. Before using the anti-static gun, ensure that the HT array plates remain in their protective pouch and placed away from the deionization area. You must place the scan tray and hybridization tray away from the area where you are performing deionization. 1. Treat the plate or lid as if it were divided into 6 sections, and deionize as follows. 2. Place a Kimwipe on the benchtop. 3. Place the stain tray on a table top. Use the anti-static gun to aim at the center of each of the six sections on a 96- well or 24-well cover or tray and pull the trigger. Ensure that a stream of ionized particles settles on all wells of the stain tray or cover to dissipate the static electricity. Squeeze and release the trigger slowly 3 times over each section (Squeeze for approximately two seconds and release for approximately two seconds). 102 GeneChip TM HT WT User Guide

104 4. Place the stain tray cover with the flat surface facing upward on the Kimwipe. Aim the anti-static gun (P/N ) approximately one-half inch away from the flat surface and pull the trigger. As you pull the trigger move the gun across the cover so that the stream of ionized particles settles on all areas of the cover and dissipates the static electricity. Squeeze and release the trigger slowly 3 times over each section (Squeeze for approximately two seconds and release for approximately two seconds). 5. Place the treated cover or tray on the Kimwipe and lift it up. 6. Do one of the following: If the Kimwipe does not adhere to the plastic, proceed with the step. If the Kimwipe adheres to the plastic, then perform steps 3 and 4 again. If it continues to adhere to the plastic, then the gun is not working and you should replace it. Figure 4.76 Removing the static charge from Stain Trays and lids. IMPORTANT: Remove static gun cover before use. If a Kimwipe sticks to treated surface, treat again following this procedure. If the Kimwipe still adheres, replace the antistatic gun. Continue the Run - Part 6: Preparation for the Gene Titan Instrument NOTE: If you are beginning the run at Part 6, click the Run button found below the menubar and make your selections from the Run Settings Options window that appears. Click OK and follow the instructions below. 1. After continuing the run from Part 5 or resuming the run by clicking the Run button and selecting run options, the Labware & Reagent Setup window appears (Figure 4.77). GeneChip TM HT WT User Guide 103

105 Figure 4.77 Part 6 Labware & Reagent Setup NOTE: If the run was stopped at the end of Part 5, manually place the sample plate at deck position P5 without a lid when ready to resume Part Setup the modular reservoir and place labware on the deck as directed in the following figures and table: Figure 4.77 illustration of the deck layout Figure 4.78 location names of empty deck positions Table 4.27 Pipette the correct reagent volumes in the Modular Reservoir. See Table 4.27 detailing the labware and Modular Reservoir placement for the deck layout. Figure 4.78 Empty Deck Positions 104 GeneChip TM HT WT User Guide

106 Table 4.27 Labware and reagent locations on the deck for Part 6: Preparation for the GeneTitan Instrument Position Labware Reagent or Samples on Deck * TL1 Biomek AP96 P250 Barrier Tips (aqua) P200-Hyb P3 Auto-sealing Microplate Lid with Microseal P Pad Lid P4 Alpaqua Magnetic Plate Magnetic Plate P5 Bio-Rad Hard Shell 96-Well PCR Plate Hyb-ready Target Hyb-ready Target from Part 5 P6 GeneTitan Hyb Tray Hybridization Tray P7 GeneTitan Stain Tray Stain 1 P8 GeneTitan Stain Tray Stain 2 P9 GeneTitan Stain Tray Stain 3 P11 GeneTitan Scan Tray Scan Tray P12 Reservoirs in frame: Quarter module reservoir in position 1, 2 and 3 Position 4 is empty Pipette Holding Buffer into reservoir 1 16 Rxn = 20 ml 24 Rxn = 20 ml 96 Rxn = 20 ml Pipette Stain 1 & 3 into reservoir 2 16 Rxn = 6 ml 24 Rxn = 6 ml 96 Rxn = entire bottle Pipette Stain 2 into reservoir 3 16 Rxn = 3 ml 24 Rxn = 3 ml 96 Rxn = 13 ml P14 Biomek Span P1000 LLS Barrier Tips (yellow) P1000-Span8 Spelt96_1 Peltier Adapter (installed on deck) * Please refer to Figure 4.78 for empty deck positions. 3. When ready to continue click Run in Figure Reagent Reservoirs Holding Buffer 4. In the prompt that appears (Figure 4.79), click Yes to begin purging the Pod2 syringe lines. Stain 1 & 3 If bubbles are still present after purging, click Purge More in the next prompt (Figure 4.79). If no further purging is required, click Skip Purge. The run proceeds. Stain 2 Empty GeneChip TM HT WT User Guide 105

107 Figure 4.79 Prompts to Purge Pod2 Syringe Lines At the completion of Part, the message shown in Figure 4.80 appears. The High Throughput Ambion WT Expression Assay for Array Plates is now complete. Figure 4.80 Message at Completion of Part GeneChip TM HT WT User Guide

108 5. Follow the instructions in the message box (Figure 4.80) 1. Discard the labware listed 2. Remove and save the following: Magnetic Plate Modular Reservoir Rack Tube Rack Span-8 tips Sample Plate 3. Preparation for GeneTitan: 1. Examine each tray to ensure that: All of the wells as appropriate (24 or 96) have been filled. If any wells do not contain reagents, then manually add reagents to these wells. There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip. 2. Carefully remove the following plates and transfer to the GeneTitan Instrument: Hybridization Tray with Target Cover and remove stain trays with stains Cover and remove scan tray with array holding buffer IMPORTANT: Immediately load the reagent plates and the Hyb Tray onto the GeneTitan Instrument. Then load the Array Plate and Hyb Tray. Do not leave samples or reagent plates at room temperature for any length of time. 4. Proceed to processing array plates using the GeneTitan Instrument as per instructions in the GeneTitan TM User Guide for Expression Array Plates (P/N ). GeneChip TM HT WT User Guide 107

109 Appendix A Using the AmbionTM 24 Rxn Kit for Two 8- Sample Runs This appendix provides instructions for using the Ambion TM WT Expression Kit for High Throughput Robotics, 24-sample reaction kit (Ambion P/N ) for two 8-sample runs. 1. Remove the following enzyme tubes and keep them on ice. First Strand Enzyme Mix Second Strand Enzyme Mix IVT Enzyme Mix Second Cycle Enzyme Mix RNase H 2. Spin the tubes in a mircofuge for 5 seconds and place them on ice. 3. Remove an aliquot from each of the tubes and put it in a new tube (already labeled and put on ice) using the information from the following table. Table A.1 Reagent Tube Size E & K Scientific Tube P/N Volume to be Aliquotted First Strand Enzyme Mix 0.5 ml μl Second Strand Enzyme Mix 0.5 ml μl IVT Enzyme Mix 0.5 ml μl Second Cycle Enzyme Mix 1.5 ml skirted μl RNase H 0.5 ml μl 4. Spin the tubes in a mircofuge for 5 seconds and place them on ice. Use this aliquot for the first 8- sample run. NOTE: Before spinning, use the lids from the original tubes to cover the newly aliquotted tubes. 5. Store the original tubes at 20 C and use these for the second 8-sample run. 6. Make sure to store the buffer mix tubes at 20 C after the first run. 108 GeneChip TM HT WT User Guide

110 Appendix B GeneChipTM HT WT Workflow Diagram This appendix provides workflow diagrams detailing the GeneChip TM HT WT workflow on the Beckman Coulter Biomek TM FX P Target Prep Express (TPE) System. The stages of the workflow are as follows: First Strand cdna Synthesis Second Strand cdna Synthesis In Vitro crna Synthesis crna Purification (diagram 1 of 8) crna Purification (diagram 2 of 8) crna Purification (diagram 3 of 8) crna Purification (diagram 4 of 8) crna Purification (diagram 5 of 8) crna Purification (diagram 6 of 8) crna Purification (diagram 7 of 8) crna Purification (diagram 8 of 8) Second Cycle cdna Synthesis - Denaturation Second Cycle cdna Synthesis - Synthesis RNase H Hydrolysis cdna Purification (diagram 1 of 8) cdna Purification (diagram 2 of 8) cdna Purification (diagram 3 of 8) cdna Purification (diagram 4 of 8) cdna Purification (diagram 5 of 8) cdna Purification (diagram 6 of 8) cdna Purification (diagram 7 of 8) cdna Purification (diagram 8 of 8) Fragmentation Labeling Cartridge Hyb Setup (diagram 1 of 2) Cartridge Hyb Setup (diagram 2 of 2) Array Plate Hyb Setup (diagram 1 of 2) GeneChip TM HT WT User Guide 109

111 Array Plate Hyb Setup (diagram 2 of 2) GeneTitan Instrument Preparation (diagram 1 of 2) GeneTitan Instrument Preparation (diagram 2 of 2) First Strand cdna Synthesis Figure B.1 First Strand cdna Synthesis 110 GeneChip TM HT WT User Guide

112 Second Strand cdna Synthesis Figure B.2 Second Strand cdna Synthesis GeneChip TM HT WT User Guide 111

113 In Vitro crna Synthesis Figure B.3 In Vitro crna Synthesis 112 GeneChip TM HT WT User Guide

114 crna Purification (diagram 1 of 8) Figure B.4 crna Purification (1 of 8) GeneChip TM HT WT User Guide 113

115 crna Purification (diagram 2 of 8) Figure B.5 crna Purification (2 of 8) 114 GeneChip TM HT WT User Guide

116 crna Purification (diagram 3 of 8) Figure B.6 crna Purification (3 of 8) GeneChip TM HT WT User Guide 115

117 crna Purification (diagram 4 of 8) Figure B.7 crna Purification (4 of 8) 116 GeneChip TM HT WT User Guide

118 crna Purification (diagram 5 of 8) Figure B.8 crna Purification (5 of 8) GeneChip TM HT WT User Guide 117

119 crna Purification (diagram 6 of 8) Figure B.9 crna Purification (6 of 8) 118 GeneChip TM HT WT User Guide

120 crna Purification (diagram 7 of 8) Figure B.10 crna Purification (7 of 8) GeneChip TM HT WT User Guide 119

121 crna Purification (diagram 8 of 8) Figure B.11 crna Purification (8 of 8) 120 GeneChip TM HT WT User Guide

122 Second Cycle cdna Synthesis Denaturation Figure B.12 Second Cycle cdna Synthesis - Denaturation GeneChip TM HT WT User Guide 121

123 Second Cycle cdna Synthesis - Synthesis Figure B.13 Second Cycle cdna Synthesis - Synthesis 122 GeneChip TM HT WT User Guide

124 RNase H Hydrolysis Figure B.14 RNase H Hydrolysis GeneChip TM HT WT User Guide 123

125 cdna Purification (diagram 1 of 8) Figure B.15 cdna Purification (1 of 8) 124 GeneChip TM HT WT User Guide

126 cdna Purification (diagram 2 of 8) Figure B.16 cdna Purification (2 of 8) GeneChip TM HT WT User Guide 125

127 cdna Purification (diagram 3 of 8) Figure B.17 cdna Purification (3 of 8) 126 GeneChip TM HT WT User Guide

128 cdna Purification (diagram 4 of 8) Figure B.18 cdna Purification (4 of 8) GeneChip TM HT WT User Guide 127

129 cdna Purification (diagram 5 of 8) Figure B.19 cdna Purification (5 of 8) 128 GeneChip TM HT WT User Guide

130 cdna Purification (diagram 6 of 8) Figure B.20 cdna Purification (6 of 8) GeneChip TM HT WT User Guide 129

131 cdna Purification (diagram 7 of 8) Figure B.21 cdna Purification (7 of 8) 130 GeneChip TM HT WT User Guide

132 cdna Purification (diagram 8 of 8) Figure B.22 cdna Purification (8 of 8) GeneChip TM HT WT User Guide 131

133 Fragmentation Figure B.23 Fragmentation 132 GeneChip TM HT WT User Guide

134 Labeling Figure B.24 Labeling GeneChip TM HT WT User Guide 133

135 Cartridge Hyb Setup (diagram 1 of 2) Figure B.25 Cartridge Hyb Setup (1 of 2) 134 GeneChip TM HT WT User Guide

136 Cartridge Hyb Setup (diagram 2 of 2) Figure B.26 Cartridge Hyb Setup (2 of 2) GeneChip TM HT WT User Guide 135

137 Array Plate Hyb Setup (diagram 1 of 2) Figure B.27 Array Plate Hyb Setup (1 of 2) 136 GeneChip TM HT WT User Guide

138 Array Plate Hyb Setup (diagram 2 of 2) Figure B.28 Array Plate Hyb Setup (2 of 2) GeneChip TM HT WT User Guide 137

139 GeneTitan Instrument Preparation (diagram 1 of 2) Figure B.29 GeneTitan Instrument Preparation (1 of 2) 138 GeneChip TM HT WT User Guide