user’s guide illumina truseq rna - perkinelmer · sciclone ngs: illumina truseq™ rna...

USER’S GUIDE ILLUMINA

TRUSEQ RNA LIBRARY PREPARATIONON THE SCICLONENGS LIQUID HANDLER

Compatible with: Illumina TruSeq™ RNA Sample Prep v2 kits Sciclone® NGS Hardware with Maestro™ 6.0 Sciclone NGSx Hardware with Maestro 6.2

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Introduction 3

Maestro TruSeq™ RNA Workflow Overview 4

Required Materials and Reagents 6

Reagents 6

General Laboratory Equipment and Supplies 6

Sciclone NGS Accessories 6

Consumables 7

Running the Maestro TruSeq™ RNA Application 7

Sample Preparation 7

Day 1: “TruSeqRNA” Run Preparation Steps 7-9

Day 2: “TruSeqDNA Library Prep” Run Preparation Steps 9-11

Day 2: PCR Enrichment 11

Day 2: “TruSeq Post-PCR SPRI” Run Preparation Steps 11-12

Library QC and Storage 12

Expected Results 13

Appendix A: Step-by-Step Guide to “TruSeqRNA” Application 13-14

Appendix B: Step-by-Step Guide to “TruSeqDNA Library Prep” Application 14-16

Appendix C: Step-by-Step Guide to “TruSeq Post-PCR SPRI” Application 16

Table of Contents

Sciclone NGS: Illumina TruSeq™ RNA

www.perkinelmer.com/ScicloneNGS 3


Introduction

Preparation of samples for mRNA sequencing on the Illumina, Inc. platform requires a series of manipulations to isolate and fragment mRNA, convert the RNA to double-stranded cDNA, and efficiently ligate appropriate indexed adapters to produce paired-end libraries. Automating the process has the advantage of avoiding sample tracking errors and reducing sample-to-sample variability while dramatically increasing throughput. The Maestro-based TruSeq™ RNA Application from PerkinElmer provides a pre-programmed solution for Illumina TruSeq™ RNA Library Preparation on the Sciclone NGS from PerkinElmer.

Figure 1. Overview of the steps for TruSeq™ RNA library preparation on the Sciclone NGS.

Maestro TruSeq™ RNA Workflow

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Maestro TruSeq™ RNA Workflow Overview

Set-up Run Time (Including Maestro Application Time Thermocycler Steps)

1 “TruSeqRNA” 1 hour 5 hours

2 “TruSeqDNA Library Prep” 1 hour 4.5 hours

3 “TruSeq Post-PCR SPRI” 10 min 45 min

Three Independent Maestro Applications are Used in the 2-day TruSeq™ RNA Workflow

The Maestro Application for TruSeq™ RNA sample preparation is a validated process for library preparation from total RNA samples that follows the steps outlined in the flowcharts on Page 5. Samples are processed in 96-well PCR plates, and the number of samples to process (1 to 12 columns of 8 samples each) is selected at the start of each run. Pre-set tip-tracking utilities written into the application guide the instrument to pick up appropriate numbers of tips and refill/replace tip boxes as needed. INHECO temperature blocks installed on the Sciclone deck allow for appropriate 4 ˚C storage of reagents and heated incubations of reactions. Reaction mixes are pre-arrayed prior to addition to sample to ensure equal incubation times across the sample plate. Adapter indexing patterns may be executed during the run by arraying 1-24 different adapters to appropriate well locations as specified by the user in an excel workbook. Easy-to-follow user interfaces guide the reagent and deck setup process and prompt the user for any necessary interventions.

The “TruSeqRNA” application method (Day 1) includes mRNA isolation and cDNA synthesis steps and follows the Illumina, Inc. protocol closely. All reactions are set up in Bio-Rad Hard-Shell® PCR plates. Washes for mRNA purification

are done with 150 µL Bead Washing Buffer (instead of the 200 µL used in the manual process). Incubations at 65 ˚C and 16 ˚C, for mRNA denaturation and 2nd strand synthesis, are done on the Sciclone deck. However, the 80 ˚C and 94 ˚C incubations for mRNA elution and fragmentation and the first-strand synthesis reactions are done in a thermocycler with a heated lid to avoid excessive evaporation. Two magnets are used in the protocol, one with a spacer to lift the plate and allow beads to pellet closer to the bottom of the wells where they are more easily resuspended by pipetting.

The Maestro “TruSeqDNA Library Prep” Application steps (Day 2) differ from the Illumina, Inc. protocol in several important aspects. First, a single aliquot of AMPure® XP beads is used per sample throughout the post-End Repair, A-tailing, and Ligation steps. The DNA in the sample is driven on and off the beads via changes in PEG and NaCl concentrations. This strategy increases yield by limiting the number of times samples are transferred to new wells/plates. Second, the volumes/concentrations of some reagent additions have been reduced from the Illumina-recommended volumes/concentrations. These reductions allow for the necessary overage volume during automated pipeting steps without significantly affecting yields. Reducing the concentration of adapters in the ligation step helps prevent excess adapter-dimer formation. Third, purification and size selection of ligation products is done with a single AMPure® XP bead cleanup, rather than gel-based size selection. The AMPure® XP cleanup is amenable to automation and high throughput sample processing, and has proved effective at reducing the adapter-dimer concentration prior to PCR enrichment of the library fragments. The modifications summarized above have led to the development of a robust automated procedure (mRNA isolation through post-PCR cleanup) for up to 96 samples that can be completed in two 6-7 hour days.

5www.perkinelmer.com/ScicloneNGS


Figure 3. Day 2 “TruSeqDNA Library Prep” and “TruSeq Post-PCR SPRI” Applications.

Figure 2. Day 1 “TruSeqRNA” Application (mRNA isolation and cDNA synthesis).

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Sciclone NGS Accessories

Accessory PerkinElmer Part No.

Agencourt 96-ring Magnet (2) CLS128316

Spacer Assembly for Agencourt CLS135277 (Qty. 4) 96-ring Magnet and CLS133514 (1)

INHECO 384-well plate adapter CLS128373

INHECO 96-well adapters (3) CLS128372

INHECO 96-well adapter/shaker CLS100852

Required Materials and Reagents

Reagents

Reagent Vendor and Part No.

TruSeq™ RNA Sample Illumina, Inc. Preparation Kit (48 samples) With Set A adapters – RS-122-2001 With Set B adapters – RS-122-2002

SuperScript II Reverse Transcriptase Invitrogen 18064-014

Nuclease-Free Water Various

AMPure® XP Beads Beckman Coulter – A63881 (60 mL)

100% EtOH Sigma-Aldrich E7023

50% PEG 8000 Sigma-Aldrich 83271

5 M NaCl Sigma-Aldrich S5150

RNase/DNase decontaminating spray Various

Note: TruSeq™ RNA kits supply enough reagents to process 48 samples. If kits are used in multiple runs with <48 samples per run, volumes of specific reagents may become limiting. Care should be taken to avoid freeze-thaw cycles with all TruSeq™ reagents. Reagents should be aliquoted appropriately if planning to use the kit for more than 2 independent runs.

Note: The Maestro “TruSeqDNA Library Prep” Application is not currently written to process samples with the Illumina-supplied End Repair Control, A-Tailing Control, or Ligase Control reagents. These kit components will not be used.

General Laboratory Equipment and Supplies

Equipment Supplier

Microfuge Various

Vortexer Various

200 µL Multichannel pipettor and appropriate barrier tips Various

1000 µL Multichannel pipettor and appropriate barrier tips Various

Microplate centrifuge Various

Thermocycler** Bio-Rad Laboratories (MJ Research) DNA Engine PTC-200, or equivalent

Plate Seals Various – compatible with Thermocycler and freezer storage

PerkinElmer LabChip® GX or Agilent Technologies Bioanalyzer with appropriate chips and reagents

** The standard sample plates used in this application are fully skirted Bio-Rad Hard-Shell® 96-PCR plates. Please check if your thermocycler is compatible with this plate type. If it is not, please contact your PerkinElmer Field Application Scientist to discuss modifications to the application to support semi-skirted PCR plates.



Consumables

PerkinElmer No. Used Consumable Description Part No. Vendor and Part No. per Run

PCR Plates 96-well PCR Plate, Bio-Rad Hard-Shell®, Full Skirt CLS127737 Bio-Rad HSP-9631 23

Boxed Tips Pipette Tip-150 µL, Art, Box, 10-96 Sterile Racks CLS111426 PerkinElmer 37 (96 samples)

Deepwell Plates Deepwell-96 POS, Square 2.0 mL Well, Polypro, CLS133355 Seahorse Bioscience 201379-100 2 Seahorse Bioscience

Reservoir- 12-Column Pyramid Bottom, 290 mL CLS128477 Seahorse Bioscience 201250-100 2 Deepwell Seahorse Bioscience

Lids 946 LID-Universal, Robotic Friendly, Polystyrene CLS112785 Seahorse Bioscience 200856-100 13

384-well Plates Microplate 384-well, Round Bottom, Polypropylene CLS134399 Corning, Inc. 3672 1

Running the Maestro TruSeq™ RNA Applications

Please read and familiarize yourself with all steps described in this section prior to beginning the run. For best results, the entire process should be completed in two 6-7 hour days. The first will include reagent preparation/distribution, deck setup, and Maestro Run for the “TruSeqRNA” application (mRNA isolation and cDNA synthesis). The second will include reagent preparation/distribution, deck setup, and Maestro Runs for the “TruSeqDNA Library Prep” and “TruSeq Post-PCR SPRI” applications.

Care should be taken to maintain an RNase-free environment during all steps of the Day 1 application. It is advisable to clean the Sciclone deck and accessories with RNase decontaminating spray prior to beginning.

Sample Preparation

Total RNA samples should be presented for the Maestro “TruSeqRNA” run in a Bio-Rad Hard-Shell® PCR plate as 0.1 to 4 µg RNA in 50 µL water. Quality of RNA samples should be checked by analysis on a LabChip GX or Agilent Technologies Bioanalyzer system: RQS or RIN scores of greater than 8.0 are required for optimal results.

The “TruSeqDNA Library Prep” application (Day 2) is optimized for 300-400 bp cDNA fragments. Samples fragmented to a smaller size will require alternative

processing (see Step 4 in “TruSeqDNA Library Prep” application steps for instructions on how to exchange AMPure® XP bead buffer to favor retention of smaller fragments in post-End Repair SPRI® cleanup).

Day 1: “TruSeqRNA” Run Preparation Steps

Note: mRNA Purification beads and AMPure® XP beads should be warmed at Room Temperature for about 30 min before use. They may be taken out of 4 ˚C storage before beginning. If frozen, TruSeq™ RNA buffers may be thawed at RT prior to beginning. (It is helpful to place the tubes containing RSB and Bead Washing Buffer in water to speed the thawing process.) Do not thaw enzyme mixes until steps 1 and 2 have been completed.

If necessary, boot up the system by first starting the Sciclone and the INHECO units, then starting the PC controller.

1. Modify the Maestro “TruSeqRNA Workbook” to Specify the Number of Samples to Run

The workbook must be located in the filepath: C:\Program Data\CaliperLS\Maestro\Workbooks\ and must have the name “TruSeqRNA Workbook.xls”. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

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The sample number must be set by indicating the number of columns to run in the worksheet titled “TruSeqRNA Reagent Setup”. The Maestro application only processes full columns of 8 samples each. Running more than 6 columns of samples will require 2 TruSeq™ RNA reagent kits, as each kit contains reagents for up to 48 samples.

After modifying the entry for the number of columns to process, the spreadsheet will update the the appropriate volumes for the reagent plates. Save the modified spreadsheet with its original name in its original file path. Print the “TruSeqRNA Reagent Setup” spreadsheet to use as a guide while setting up reagents.

2. Start the Maestro “TruSeqRNA” Application

Launch the Maestro software and open the “TruSeqRNA” Application. Start the run by selecting the play button. If running in Editor mode, be sure to start the Main Method.

Note: When the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to proceeding with the run. Starting the application prior to thawing and diluting reagents ensures that the cold blocks are pre-chilled and ready for on-deck reagent storage.

Ensure that the INHECO units have the correct adapters for the “TruSeqRNA” Application Position A3 96-well PCR Plate Adapter Position A4 96-well PCR Plate Adapter Position D2 96-well PCR Plate Adapter Position D4 96-well PCR Plate Shaker Adapter

Verify that the INHECO units for positions A3 and A4 are set to 4 ˚C and are cooling. Verify that the INHECO unit for position D4 is set to 25 ˚C and the INHECO unit for position D2 is set to 65 ˚C and is heating.

3. Thaw the Enzyme Mixes and Place on Ice

First Strand Master Mix, Second Strand Master Mix, and SuperScript II should be thawed at this time. Care should be taken to avoid freeze-thaw cycles with all enzyme mixes. Reagents should be aliquoted appropriately if planning to use a kit for more than 2 independent runs.

4. Prepare the Buffer, Bead and 80% EtOH Plates

Using a multichannel pipettor, aliquot the indicated volumes of buffers to the Bio-Rad Hard-Shell® PCR plates and wells specified on the spreadsheet. Plates should be made for: Bead Binding Buffer, Bead Washing Buffer (2), Elution Buffer, Resuspension Buffer, and Elute/Prime/Fragment Mix. Label the plates, cover, and store at room temperature until ready to set up the Sciclone deck.

Thoroughly resuspend mRNA purification beads and AMPure® XP beads (warmed to room temperature) by inverting/rotating the tube/bottle. Using a multichannel pipettor, aliquot the indicated volumes of beads to the specified wells of Bio-Rad Hard-Shell® PCR plates. Label the plates, cover, and store at room temperature until ready to set up the Sciclone deck.

Inspect all plates to ensure that air has not been trapped in the wells. If necessary, spin the plates briefly to bring reagents to the bottom of the wells.

Make 100 mL fresh 80% EtOH solution by diluting 80 mL 100% Ethanol with 20 mL nuclease-free molecular biology grade water. Pour 100 mL fresh 80% EtOH into a Seahorse Bioscience deepwell reservoir, cover with a lid and store at room temperature.

5. Prepare the Enzyme Reagent Plate and Sample Plate

Prepare First Strand Master Mix by adding 50 µL SuperScript II to the Illumina supplied tube. Mix gently by inverting and spin down. If running fewer than 48 samples, label the tube to indicate that the SuperScript II has been added prior to storing the tube at -20 ˚C.

Gently mix and spin the Second Strand Master Mix tube.

Fill the specified columns of a Bio-Rad Hard-Shell® PCR plate with the specified volumes of First Strand and Second Strand Master Mixes. Keep the plate on ice while pipetting to keep the reagents cold. Pipet carefully into the bottom of the wells and avoid trapping air or creating bubbles. If necessary, spin the plates briefly in a plate centrifuge to ensure all reagents are at the bottom of the wells. Label the plate, cover with a lid and store on ice or at 4 ˚C.

Figure 4. “TruSeqRNA Reagent Setup” Spreadsheet.



6. Set up the Sciclone Deck

Confirm that the Maestro software has correctly read the workbook and is set to run the correct number of columns. If the incorrect number of columns is indicated, modify and save the excel workbook as described in Step 1, then start the application again from the Main Method.

Step through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

7. Run mRNA Isolation and cDNA Preparation Steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the protocol.

The Maestro “TruSeqRNA” Application will automatically proceed through the steps indicated in the flowchart in Figure 2 (Page 5). While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. See Appendix A (Page 12) for a Step-by-Step guide to the Sciclone steps of the Maestro “TruSeqRNA” Application.

When it is necessary to transfer the samples to a thermocycler, the application will pause and show a message indicating that the PCR plate should be sealed and run on the thermocycler with the appropriate program. It is advised to pre-program the thermocycler with methods for mRNA Elution, Elution/Frag/Prime, and 1st Strand as described in the Illumina, Inc. TruSeq™ RNA protocol:

mRNA Elution 1 80 ˚C for 2 min (lid at 100 ˚C) 25 ˚C hold

Elution 2 – Frag – Prime 94 ˚C for 8 min (lid at 100 ˚C) 4 ˚C hold

1st Strand Run with lid at 100 ˚C 25 ˚C for 10 min 42 ˚C for 50 min 70 ˚C for 15 min 4 ˚C hold

Note: Incubations for mRNA Denaturation and Second Strand synthesis will be done on the Sciclone deck.

Note: Please ensure that your thermocycler is compatible with fully skirted PCR plates.

8. Store cDNA samples

When the application is complete, you will be prompted to seal the plate containing cDNA in 50 µL RSB for storage at -20 ˚C overnight. After storing the plate, close the dialog box. The Sciclone will shut off the leg lights and inactivate the INHECO temperature control.

Day 2: “TruSeqDNA Library Prep” Run Preparation Steps

Note: AMPure® XP beads should be warmed at Room Temperature for about 30 min before use. They may be taken out of 4 ˚C storage before beginning. If frozen, RSB buffer may be thawed at RT prior to beginning. (It is helpful to place the tube containing RSB in water to speed the thawing process.) Do not thaw other reagent mixes until Steps 1 and 2 have been completed.

If necessary, boot up the system by first starting the Sciclone and the INHECO units, then starting the PC controller.

1. Modify the Maestro “TruSeqDNA Library Prep Workbook” to Specify the Number of Samples to Run and the Adapter Index Pattern

The workbook must be located in the filepath: C:\Program Data\Caliperls\Maestro\Workbooks\ and must have the name “TruSeqDNA Library Prep Workbook.xls”. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

The sample number must be set by indicating the number of columns to run in the worksheet titled “TruSeq DNA Reagent Plates”. The Maestro application only processes full columns of 8 samples each. Running more than 6 columns of samples will require 2 TruSeq™ DNA reagent kits, as each kit contains reagents for up to 48 samples.

The adapter index pattern is set by designating the appropriate adapter numbers to each well ID in spreadsheet titled “Indexing”. Up to 24 different adapters may be used. Illumina, Inc. supplies 20 µL per tube of each adapter, allowing up to 8 samples per adapter when diluting adapter 1:2 with RSB.

Figure 5. “TruSeqDNA Reagent Plates” Spreadsheet in the TruSeq DNA Library Prep Workbook.

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Enter a number from 1-27 in the “Index Well” column for each of the samples to be run. The TruSeq™ adapters are numbered from ID001 to ID027, with adapters ID017, ID024 and ID026 omitted. If desired, custom primers can be used and their names inputted in cells I4-I30 of the Indexing spreadsheet. The chart on the right will automatically update the number of samples using each adapter index. This information will be passed into the worksheet for “TruSeqDNA Reagent Plates” to modify the volume necessary for each adapter index mix.

After modifying entries for the number of columns to process and the adapter index pattern, the spreadsheet will update the recipes for the reagent mixes and the appropriate volumes for the reagent plates.

Save the modified spreadsheet with its original name in its original file path. Print the “TruSeqDNA Reagent Plates” spreadsheet to use as a guide while setting up reagents.

2. Start the Maestro “TruSeqDNA Library Prep” Application

Launch the Maestro software and open the “TruSeqDNA Library Prep” Application. Start the run by selecting the play button. If running in Editor mode, be sure to start the Main Method.

Note: when the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps. Starting the application prior to thawing and diluting reagents ensures that the cold blocks are pre-chilled and ready for on-deck reagent storage.

Ensure that the INHECO units have the correct adapters for the “TruSeqDNA Library Prep” Application. Verify that the INHECO units for positions A3, A4 are set to 4C and are cooling.

Position A3 384-well Plate AdapterPosition A4 96-well PCR Plate AdapterPosition D2 96-well PCR Plate AdapterPosition D4 96-well PCR Plate Shaker Adapter

3. Thaw the TruSeq™ DNA Reagents and Place on Ice

Care should be taken to avoid freeze-thaw cycles with all TruSeq™ DNA reagents. Reagents should be aliquoted appropriately if planning to use a kit for more than 2 independent runs. It is helpful to place the tubes containing RSB and End Repair Mix in water to speed the thawing process.

4. Prepare the 80% EtOH Reservoir, PEG Plate, RSB Plate, and AMPure® XP Bead Plate

Make 100 mL fresh 80% EtOH solution by diluting 80 mL 100% Ethanol with 20 mL nuclease-free molecular biology grade water. Pour 100 mL fresh 80% EtOH into a Seahorse Bioscience deepwell reservoir, cover with a lid and store at room temperature.

Use the “TruSeqDNA Reagent Plates” spreadsheet as a guide for setting up the plates.

If necessary, make a 20% PEG/2.5M NaCl solution and 20% PEG/3M NaCl solution. These solutions may be made in advance and stored at room temperature.

Using a multichannel pipettor, aliquot 30 µL 20% PEG/2.5M NaCl solution per well into a Bio-Rad Hard-Shell® PCR plate for each column of samples to be run. Label the plate, cover with a lid, and store at room temperature.

Using a multichannel pipettor, aliquot 120 µL Resuspension Buffer per well into a Bio-Rad Hard-Shell® PCR plate for each column of samples to be run.

Note: If it is desired to retain smaller fragments (150 to 250 bp) in the post-ER SPRI cleanup, then AMPure® XP beads will need to undergo a bead buffer exchange step to substitute the existing buffer on the AMPure® XP beads with an equal volume of 20% PEG/3M NaCl solution. The higher salt content in the bead buffer will allow recovery of shorter fragments (<250 bp) in the post End Repair SPRI® cleanup step.

Figure 6. “Indexing” spreadsheet in the “TruSeqDNA Library Prep Workbook”.

Figure 7. “TruSeqDNA Reagent Plates” Spreadsheet in the “TruSeqDNA Library Prep Workbook”.



To exchange the bead buffer, thoroughly resuspend AMPure®

XP beads (warmed to room temperature) by inverting/rotating the bottle. Transfer the total volume of beads needed (plus 10% overage volume) to one or more 2 mL eppendorf tubes or to a 15 mL falcon tube. Pellet the beads using a magnet. Remove the buffer from the beads and replace it with an equal volume of 20% PEG/3M NaCl. Thoroughly resuspend the beads by inverting/rotating the tube(s).

Using a multichannel pipettor, aliquot 105 µL AMPure® XP beads per well into a Bio-Rad Hard-Shell® PCR plate for each column of samples to be run. Cover the plate with a lid and store at room temperature.

Inspect all plates to ensure that air has not been trapped in the wells. If necessary, spin the plates briefly to bring reagents to the bottom of the wells.

5. Make the Reaction Master Mixes and Adapter Mixes

The following mixes should be prepared according to the recipes in the “TruSeqDNA Reagent Plates” spreadsheet: End-Repair Mix, A-Tailing Mix, Ligation Mix, Adapter Mixes (up to 24), PCR Mix.

Gently mix and centrifuge each Illumina supplied reagent tube prior to use. After mixing the appropriate volumes of reagents in nuclease-free tubes, mix by inverting and centrifuge again to collect all liquid at the bottoms of the tubes.

Care should be taken to pipet accurately, as some reagents are viscous and minimal overage volumes are used. Keep the reaction mixes on ice.

6. Aliquot the Reaction Mixes into Plates

The Bio-Rad Hard-Shell® PCR plate should be filled with the specified volumes of A-tailing Mix, Ligase Mix, Stop Ligase Buffer, Adapter Mixes, End Repair Mix and PCR Master Mix in the specified wells. Aliquot the reaction mixes to the reagent plate in the wells indicated on the “TruSeqDNA Reagent Plates” spreadsheet. Keep the plates on ice while pipetting to keep the reagents cold. Pipet carefully into the bottom of the wells and avoid trapping air or creating bubbles. If necessary, spin the plate briefly in a plate centrifuge to ensure all reagents are at the bottom of the wells. Label the plate, cover with a lid and store on ice or at 4 ˚C.

7. Set up the Sciclone Deck

Confirm that the Maestro software has correctly read the workbook and is set to run the correct number of columns. If the incorrect number of columns is indicated, modify and save the excel workbook as described in Step 1, then start the application again from the Main Method.

Step through the pictures, placing the indicated consumables/ prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

8. Run Library Preparation Steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the library preparation protocol.

The Maestro “TruSeqDNA Library Prep” application will automatically proceed through End Repair, A-tailing, Ligation, and PCR setup steps as indicated in the flowchart (Figure 2, Page 5). While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. See Appendix B (Page 13) for a Step-by-Step guide to the Sciclone steps of the “TruSeqDNA Library Prep” application.

When the PCR setup is complete, the application will pause and show a message indicating that the PCR plate should be sealed and placed on a thermocycler for the amplification step. Close the dialog box to complete the run and shut down the leg lights and INHECO temperature controls.

Note: The RSB and 80% EtOH plates may be retained for use in the Post-PCR SPRI® cleanup.

Day 2: PCR Enrichment

Illumina, Inc. recommends the following program for amplification of libraries prepared from 0.1 to 4 µg total RNA. The number of cycles may need to be modified according to the amount and type of input RNA in the sample. Use a heated lid to prevent condensation.

PCR:

98 ˚C for 30 secs.15 cycles of:

98 ˚C for 10 secs.60 ˚C for 30 secs.72 ˚C for 30 secs.72 ˚C for 5 mins.Hold at 10 ˚C

Day 2: “TruSeq Post-PCR SPRI” Run Preparation Steps

The “TruSeq Post-PCR SPRI” cleanup step is provided as a separate Maestro application. If desired, the user may designate a different liquid handler for post-PCR sample processing to avoid any possible cross-contamination of pre-amplification samples. “TruSeq Post-PCR SPRI” cleanup applications are available for both the Sciclone NGS and the Zephyr workstations. The instructions here are for running the “TruSeq Post-PCR SPRI” application on the Sciclone.

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Note: AMPure® XP beads should be warmed at Room Temperature for about 30 min before use. They may be taken out of 4 ˚C storage before beginning. If the RSB remaining in the Bio-Rad Hard-Shell® 96-well plate has not been saved at the completion of the library prep, it will also be necessary to thaw the RSB buffer from the Illumina, Inc. library prep kit.

1. Open the “TruSeq Post-PCR SPRI Workbook”

Enter the number of columns to be processed and save the workbook. The workbook must be located in the filepath: C:\ProgramData\Caliperls\Maestro\Workbooks\ and must have the name “TruSeq Post-PCR SPRI Workbook.xls”. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

2. Start the Maestro “TruSeq Post-PCR SPRI” Application

Launch the Maestro software and open the “TruSeq Post-PCR SPRI” application. Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method.

Note: When the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps.

3. Prepare the SPRI® Beads Plate and RSB Plate

Aliquot 55 µL of AMPure® XP beads per well in a Bio-Rad Hard-Shell® PCR plate as indicated in the “TruSeq Post-PCR SPRI Workbook”. Ensure that the beads are at room temperature and well mixed prior to pipetting.

If the 96-well Bio-Rad Hard-Shell® PCR plate containing RSB used in the “TruSeqDNA Library Prep” application is saved at the completion of the library prep steps, the same plate may be used for RSB during the post-PCR cleanup. Confirm that enough RSB remains in the wells (30 µL plus >5 µL overage volume in each well), or set up a new plate as indicated in the workbook.

4. Prepare 80% Ethanol Reservoir

If the 80% EtOH reservoir used in the “TruSeqDNA Library Prep” run has been retained, it can be used for this run. Otherwise, make 50 mL fresh 80% EtOH solution by diluting 40 mL 100% Ethanol with 10 mL nuclease-free molecular biology grade water. Pour 50 mL fresh 80% EtOH into a Seahorse Bioscience deepwell reservoir and cover with a lid.

5. Setup the Sciclone Deck

Confirm that the software has correctly read the workbook and is set to run the correct number of columns. Step through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

Note: when the “TruSeq Post-PCR SPRI” application is started, the variables used for tip tracking are reset. The run must be started with new, full tip boxes in the indicated positions, as Maestro will not retain tip tracking information from the library prep run.

6. Run the “TruSeq Post-PCR SPRI” Steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the library prep steps. See Appendix C for a Step-by-Step guide to the Sciclone steps of the “TruSeq Post-PCR SPRI” application.

When the run is complete, the application will pause and show a message indicating that the PCR plate should be sealed and stored appropriately. Close the dialog box to complete the run and shut down the leg lights and INHECO temperature controls.

Library QC and Storage

Seal the library plate and store at -20 ˚C for up to 7 days, or proceed directly into library validation prior to storing.

The LabChip GX may be used to check the size distribution of fragments in the amplified library and estimate the concentration of fragments in the appropriate size range. Make a 1:25 dilution of library into molecular biology grade water in a Bio-Rad Hard-Shell® skirted 96-PCR plate. Mix well by pipeting up and down, and spin the plate to remove any bubbles. Run the samples on the GX using a High Sensitivity DNA chip and kit according to the standard LabChip protocol.

Additional/alternative validation and quantification, including qPCR quantification, should be done according to the user’s standard practices.

Figure 8. “SPRI Reagents” Spreadsheet in the “TruSeq Post-PCR SPRI” Workbook.



Expected Results Appendix A

Step-by-Step Guide to “TruSeqRNA” Application (Day 1 mRNA Isolation and cDNA Synthesis)

Step 1: Oligo dT Capture

1. Load tips. Transfer 50 µL sample (B4) to 50 µL mRNA beads (D4) and mix.

2. Dispose of sample plate.

3. Move sample/beads to 65 ˚C (D2) for 5 min, 4 ˚C (A4) for 1 min, then RT (D4) for 5 min.

4. Move plate to magnet with spacer, incubate 2 min, remove supernatant to waste.

5. Move bead plate to D4, dispose of tips.

6. Move Bead Wash Buffer from A2 to B4, load tips, transfer 150 µL Bead Wash Buffer (B4) to beads at D4, mix, shake 1 min.

7. Dispose of Bead Wash Buffer Plate (B4).

8. Mix beads and Wash Buffer at D4.

9. Move plate to magnet with spacer, incubate 2 min, remove supernatant to waste.


11. De-lid Elution Buffer plate (B2) and move to B4. Replace lid at B2.

12. Load tips, transfer 50 µL Elution Buffer (B4) to beads at D4, mix, shake 1 min.

13. Dispose of Elution Buffer plate, dispose of tips.

14. Seal plate, spin if necessary, transfer to off-deck thermocycler, run mRNA Elution 1 with heated lid.

15. Unseal bead plate, place on D4 with lid.

16. Move Binding Buffer from A2 to B4

17. Load tips, transfer 50 µL Binding Buffer (B4) to beads (D4), mix, shake 1 min.

18. Dispose of Binding Buffer plate (B4).

19. Incubate samples/beads 5 min at RT (D4).

20. Move plate to magnet with spacer (C4), incubate 2 min, remove supernatant to waste.

Figure 9. LabChip GX data from a 1:25 dilution of library prepared from 1 µg total RNA (Human Reference Brain RNA, LifeTech, #AM6051).

Figure 11. Data based on LabChip GX smear analysis of 1:25 dilutions of libraries shown in Figure 10.

Figure 10. LabChip GX data from 1:25 dilutions of libraries prepared from replicate samples at 3 different input concentrations (4 µg, 1 µg, and 0.2 µg Human Reference Brain RNA, LifeTech, #AM6051).

14



22. Move Bead Wash Buffer from A2 to B4

23. Load tips, transfer 150 µL Bead Wash Buffer (B4) to beads at D4, mix, shake 1 min.

24. Dispose of Bead Wash Buffer Plate (B4).

25. Mix beads and Wash Buffer at D4.

26. Move plate to magnet with spacer (C4), incubate 2 min, remove supernatant to waste.


28. De-lid and move EFP plate (C2) to B4. Replace lid on SPRI® plate at C2.

29. Load tips, transfer 19.5 µL EFP (B4) to beads (D4), mix, shake 1 min.

30. Dispose of EFP plate (B4).

31. Mix beads and EFP (D4), dispose of tips.

32. Seal plate, spin if necessary, transfer to off-deck thermocycler, and run Elution2 – Frag – Prime program with heated lid.

Step 2: First Strand Synthesis

1. Unseal bead plate, place on magnet on B4 with lid. Incubate for 5 mins.

2. Move clean plate (D3) to A4.

3. Load tips (single column), broadcast 1st strand mix from column 1 Reagent plate (A3) to plate at A4, dispose of tips.

4. Load tips, transfer supernatant from beads on magnet (B4) to 1st strand mix at A4, mix, dispose of tips.

5. Move 1st strand reaction to D4, shake 1 min, dispose of EFP/bead plate and lid (B4).

6. Seal plate, spin if necessary, transfer to off-deck thermocycler, and run 1st Strand program with heated lid.

Step 3: Second Strand Synthesis

1. Unseal plate and place on shaker at D4, no lid.

2. Move active tip box (C3) out of way to A2, move clean plate (D3) to A4, move active tip box back to C3.

3. Load tips (single column), broadcast 2nd Strand Master Mix from Reagent plate A3 to clean plate at A4, dispose of tips.

4. Load tips, transfer 25 µL 2nd strand mix from A4 to samples at D4, mix, dispose of tips cover with lid from A3.

5. Dispose of plates at A4 and A3.

6. Incubate 2nd Strand reaction at 16 ˚C (D4) for 60 min.

Step 4: SPRI® Cleanup

1. Dispose of lid from sample plate (D4) and lid from SPRI® beads plate (C2).

2. Move SPRI® beads plate (C2) to A4.

3. Load tips, resuspend beads by mixing at A4.

4. Transfer 90 µL beads to samples at D4, mix, incubate with shaking 10 min at RT.

5. Move samples/beads to magnet at B4, incubate 6 min, remove SN to waste, dispose of tips.

6. Move lid from 80% EtOH (B5) to empty tip box (C5).

7. Load tips, wash beads with 150 µL 80% EtOH, remove EtOH to waste (A5), dispose of tips. Repeat.

8. Move beads (B4) to 37 ˚C (D2) to dry. Incubate 10 min.

9. Move beads (D2) to RT (D4).

10. Load tips, transfer 52 µL RSB to beads, mix, incubate 5 min at RT.

11. Dispose of RSB plate at A4.

12. Mix beads at D4, dispose of tips.

13. Move beads/sample (D4) to magnet at B4, incubate 5 min.

14. Move tip box at C3 out of way to A2, move clean plate D3 to A4, Move tip box back to C3.

15. Load tips, transfer 50 µL samples from beads (B4) to clean plate (A4).

16. Move sample plate to D4.

17. Show application finished dialog.

Appendix B

Step-by-Step Guide to “TruSeqDNA Library Prep” Application (Day 2)

Step 1: Broadcast End Repair

1. Move lid on Reagent Plate A4 to C4

2. Load tips (single column) and transfer 45 µL of ERP from Col 8-10 of the Reagent Plate (A4) to the PCR Plate on CPAC in D2, Eject tips.

3. Move lid C4 to Reagent Plate A4.

Step 2: End Repair

1. Move lid from Sample Plate (D4) to C4.

2. Load tips. Transfer 40 µL ERP (D2) to sample plate (D4), Mix, Eject tips.

3. Dispose of ERP plate (D2).

4. Move lid from C4 to Sample Plate D4.



5. Incubate End Repair Reaction for 30 mins at 30 ˚C.

During Incubation Pre-Broadcast Adapters to 384-well Plate

a. Move lid A4 to C4. Move lid A3 to A2.

b. Load tips (single column) and eject to empty tip box at C5. Load single tip from C5 for each adapter transfer.

c. Broadcast adapters one at a time. Aspirate from Reagent Plate A4 col 4-6 and multi-dispense to all appropriate wells in Q1 of 384-well at A3. Eject tip. Repeat for all adapters.

d. Move lid C4 to Reagent Plate (A4). Move lid A2 to 384-well Plate (A3).

6. Remove Lid from Sample Plate (D4) to A2. Move Sample Plate from D4 to C4.

7. Dispose of Lid on 2 PCR Plates at B2. Move top plate (SPRI® Bead Plate) from B2 to D4.

8. Remove any remaining tips from Tip Box in C5 and Eject to Waste.

9. Load tips. Mix Beads at D4. Transfer 100 µL of beads from D4 to Sample Plate at C4. Eject Tips.

10. Dispose of used Bead Plate (D4).

11. Move Sample Plate from C4 to D4. Load tips and Mix at D4.

12. Move lid from A2 to Sample Plate (D4).

13. Incubate 10 mins

During Incubation, Pre Broadcast ATL to 384-well Plate

a. Move lid A3 to B2. Move lid A4 to C4.

b. Load tips (single column).

c. Transfer 15 µL A-Tail Mix from Col 1 of Reagent Plate (A4) to Q2 of 384-well Plate (A3). Repeat until broadcast is complete. Eject Tips.

d. Move lid B2 to 384-well Plate (A3). Move lid C4 to Reagent Plate (A4).


15. Move Sample Plate (D4) to Magnet (B4). Incubate 5 min.

16. Load tips. Remove 90 µL supernatant from Sample Plate (B4) and dispense to liquid waste (A5). Repeat. Eject Tips

17. Move lid C2 is moved to A2.

18. Move lid from 80% EtOH (B5) to empty tip box (C5).

19. Load tips, wash beads with 150 µL 80% EtOH, remove EtOH to waste (A5), dispose of tips. Repeat.

20. Move lid from C5 back to B5

21. Move beads (B4) to 37 ˚C (D2) to dry. Incubate 10 min.

22. Move beads (D2) to RT (D4).

23. Load tips, transfer 15 µL RSB (C2) to beads (D4), mix, incubate 3 min at RT.

24. Mix for 3 more cycles after shaking is complete. Eject tips

25. Move lid A2 to RSB plate (C2).

Step 3: A-Tailing

1. Move lid from 384-well Plate (A3) to B2.

2. Load tips. Transfer 15 µL ATL from Q2 of 384-well plate (A3) to sample plate D4. Mix. Eject Tips.

3. Move lid from B2 to 384-well Plate (A3).

4. Incubate for 30 min at 37 ˚C at D4.

During Incubation, Pre-Broadcast Ligase Mix to 384-well Plate

a. Move lid A3 to B2. Move lid A4 to C4.


c. Transfer 8ul Ligase Mix from Col 2 of Reagent Plate (A4) to Q4 of 384-well Plate (A3). Repeat until broadcast is complete. Eject Tips.



Step 4: Ligate Adapters

1. Move lid from 384-well Plate (A3) to B2.

2. Load tips. Aspirate 3.5 µL of adapter from Q1 of 384-well plate (A3) and dispense 1 µL back to plate (A3). Dispense 2.5 µL to Sample Plate (D4).

3. Transfer 5 µL ligase mix (2 step aspirate) from Q4 of 384-well plate (A3) to Sample Plate (D4). Mix. Eject Tips.

4. Move lid C4 to Sample Plate (D4).

5. Incubate for 10 min at 30 ˚C

During Incubation, Pre Broadcast STL to 384-well Plate

a. Move lid A4 to C4.


c. Transfer 8 µL STL from Col 3 of Reagent Plate (A4) to Q3 of 384-well plate (A3). Repeat until broadcast is complete. Eject Tips.


6. Remove lid from Sample Plate (D4) to C4.

7. Move lid from 384-well Plate (A3) to B2

8. Load tips. Transfer 5 µL of STL (2 step aspirate) from Q3 of 384-well plate (A3) to Sample Plate (D4). Mix. Eject Tips.

9. Move lid B2 to 384-well Plate (A3)

10. Load tips. Transfer 12.5 µL PEG from B2 to Sample Plate (D4). Mix. Eject Tips.

11. Move Lid C4 to Sample Plate (D4).

12. Incubate for 10 mins.

13. Remove lid from Sample Plate (D4) to C4.

14. Move Sample Plate (D4) to Magnet (B4)

15. Incubate for 10 mins.

During Incubation, Pre Broadcast PMM to PCR Plate

a. Load tips (single column).

b. Move Reagent Plate and Lid (A4) to D2.

c. Move top plate (of stack of 3) from D3 to A4.

d. Move lid from Reagent Plate (D2) to A2.

e. Transfer 30 µL from Col 11-12 of Reagent Plate (D2) to PCR Plate (A4). Repeat until broadcast is complete. Eject Tips.

f. Move lid A2 to Reagent Plate (D2).

g. Dispose of Lid from D2. Dispose of plate from D2.

16. Move Lid from C4 to PCR Plate (A4)

17. Load tips. Transfer 45 µL Supernatant from B4 to Liquid Waste (A5). Eject Tips

18. Move lid from RSB plate (C2) to A2.

19. SPRI® Clean Up (same as steps 18-21 under Step 2 End Repair)

20. Load tips, transfer 20 µL RSB (C2) to beads (D4), mix, incubate 5 min at RT.

21. Move Lid back to Resuspension Buffer Plate (C2) from A2.

Step 5: Setup PCR

1. Move Sample Plate (D4) to Magnet (B4). Incubate 5 mins.

2. Move lid from PCR Plate (A4) to C4.

3. Load tips. Transfer 20 µL from Sample Plate (B4) to PCR Plate (A4). Mix. Eject tips.

4. Move lid from C4 to PCR Plate (A4)

5. Move Head to A1. Show Application Complete Dialog.

Appendix C

Step-by-Step Guide to “TruSeq Post-PCR SPRI” Application (Day 2)

1. Load tips. Transfer 50 µL sample from PCR plate on magnet at B4 to clean PCR plate at D4. Eject tips.

2. Load tips. Mix AMPure® XP beads at C2. Transfer 50 µL beads from C2 to samples at D4. Mix.

3. Incubate beads and sample for 10 min. Dispose of bead plate at C2 and PCR plate at B4.

4. Move beads/sample from D4 to magnet at B4, incubate 10 mins.

5. Remove 100 µL supernatant to waste at A5. Eject tips.

6. Remove lid from 80% EtOH reservoir at B5 and place on empty tip box at C5.

7. Wash beads with 150 µL 80% EtOH two times. Replace lid on 80% EtOH reservoir.

8. Move beads to D2 to dry at 37 ˚C for 5 mins.

9. Move beads from D2 to D4.

10. Move lid from RSB at C4 to clean plate at C2.

11. Transfer 32 µL RSB from C4 to beads at D4. Mix. Eject tips.

12. Transfer sample/beads (D4) to magnet at B4, incubate 5 mins.

13. Move lid from C2 to C4. Move clean PCR plate from C2 to A4.

14. Transfer 30 µL sample from beads at B4 to clean plate at A4.

15. Move sample plate from A4 to D4 (4 ˚C). Move lid from C4 to D4.

16. Move head out of way and show application complete dialog.

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Copyright ©2012, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 010460_01 Printed in USA Aug 2012

PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602www.perkinelmer.com


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