SureSelect RNA Target Enrichment for Illumina Paired
Contents
1. Reagent Kits 16 Reactions 96Reactions 480 Reactions SureSelect TE RNA Reagent Kit HSQ G9601A G9601B G9601C Table 4 SureSelect Capture Library select one Capture Libraries 16 Reactions 96 Reactions 480 Reactions SureSelect RNA Kinome 5190 4801 5190 4802 5190 4803 SureSelect RNA Capture 1 kb up to 499 Kb 5190 4934 5190 4935 5190 4937 reorder 5190 4939 5190 4940 5190 4942 SureSelect RNA Capture 0 5 Mb up to 2 9 Mb 5190 4944 5190 4945 5190 4947 reorder 5190 4949 5190 4950 5190 4952 SureSelect RNA Capture 3 Mb up to 5 9 Mb 5190 4954 5190 4955 5190 4957 reorder 5190 4959 5190 4960 5190 4962 10 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Before You Begin 1 Table 5 Required Reagents for Hybridization Description Vendor and part number Dynabeads MyOne Streptavidin T1 Life Technologies 2mL Cat 65601 10mL Cat 65602 100 mL Cat 65603 Nuclease free Water not DEPC treated Ambion Cat AM9930 Optional Reagents Table 6 Optional Reagents Description Vendor and part number Ethylene glycol American Bioanalytical p n AB00455 RNeasy MinElute Cleanup Kit Qiagen p n 74204 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 11 1 12 Before You Begin Required Equipment Table 7 Required Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number 2100 Bioanalyzer Agilent p n G2938C2. and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Right
3. 4 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Content 1 Before YouBegin 7 Procedural Notes 8 Safety Notes 8 Required Reagents 9 Optional Reagents 11 Required Equipment 12 Optional Equipment 14 2 Sample Preparation 15 Step 1 Purify the mRNA 18 Step 2 Fragment the RNA 18 Step 3 Clean up the fragmented mRNA 19 Step 4 Synthesize first strand cDNA 20 Step 5 Synthesize second strand cDNA 22 Step 6 Purify the sample with the QlAquick PCR Purification Kit 23 Step 7 Repair the ends 24 Step 8 Purify the sample with the QlAquick PCR Purification Kit 26 Step 9 Add dA Bases to the 3 end of the cDNA fragments 27 Step 10 Purify the sample with the QlAquick PCR Purification Kit 28 Step 11 Ligate the paired end adaptor 29 Step 12 Purify the sample with the QlAquick PCR Purification Kit 30 Step 13 Size select the DNA fragments with a E Gel SizeSelect 2 Agarose gel 31 Step 14 Purify the sample with the QlAquick PCR Purification Kit 33 Step 15 Amplify adaptor ligated library 34 Step 16 Purify the sample with the QlAquick PCR Purification Kit 37 Step 17 Assess quality and quantity with 2100 Bioanalyzer 38 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Contents 3 Hybridization 41 Step 1 Hybridize the library 44 Step 2 Prepare magnetic beads 50 Step 3 Select hybrid capture with SureSelect 51 Step 4 Purify the
4. see Figure 2 6 Collect the solution from the sample well 7 Wash each collection well with 25 uL of nuclease free water pipette up and down then retrieve the wash solution and combine with the respective sample solution collected in step 6 for a total of 50 uL See Table 21 for expected lengths of the insert and PCR according to the excised cDNA length SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 31 2 Sample Preparation Sample wells 250 bp Collection wells 150 bp Figure 2 Elution of an approximately 200 bp region This image shows three samples on the same gel Table 21 Expected lengths of the insert and PCR according to the excised cDNA length Excised cDNA length nt Insert length bp PCR product length bp 50 0 100 100 50 150 150 100 200 200 150 250 250 200 300 32 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 14 Purify the sample with the QlAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit Qiagen p n 28104 1 If you haven t already done so add the pH Indicator I to the Buffer PB 2 Add 250 uL of Buffer PB to 50 uL of sample and mix well by pipetting Check for the yellow color to make sure Buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen QIAquick Handbook If needed use 5 uL of the 3M Sodium Acetate incl
5. C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 13 Size select the DNA fragments with a E Gel SizeSelect 2 Agarose gel Use the E Gel SizeSelect 2 Agarose Gel 1 Remove a E Gel SizeSelect 2 Agarose Gel from its package Remove the combs from the top sample loading wells and the middle collection wells Set the E Gel on the E Gel iBase linked with the E Gel Safe Imager 2 Load the E Gel as follows a Load 20 uL of the ligated purified DNA into a well in the top row Do not use the center well or outermost wells to avoid edge effects Do not load more than 1 ug of DNA If the sample volume lt 20 uL add nuclease free water to the well for a total volume of 20 uL b Load 2 uL 50 bp ladder at 0 1 ug uL to the center top well Add 15 uL of water to fill the well c Fill empty wells in the top row with 20 uL of nuclease free water d Fill each of the collection wells in the middle of the gel with 25 uL of nuclease free water Add 20 uL of nuclease free water to the middle center well 3 Run the gel iBase program Run E Gel DC Approximate run time 13 45 13 minutes and 45 seconds Monitor the E Gel in real time with the E Gel Safe Imager 4 If needed during the run fill the middle collection wells with nuclease free water 5 When the 200 bp band from the marker lane is in the center of the collection well stop the run if the run has not already stopped
6. Il Fusion DNA Polymerase Agilent Component DMSO green cap 5X Herculase Il Rxn Buffer clear cap 100 mM dNTP Mix green cap Herculase II Fusion DNA Polymerase red cap Table 36 D1K Reagents Agilent p n 5067 5362 Components ladder D1K sample buffer Table 37 High Sensitivity D1K Reagents Agilent p n 5067 5364 Components High Sensitivity D1K ladder High Sensitivity D1K sample buffer Table 38 TruSeq Cluster Generation Kit Components HT1 Hybridization Buffer HP3 2 N NaOH Use the Illumina Cluster Generation Kit that is appropriate for your instrument and setup See Table 5 on page 11 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Reference 5 Table 39 PhiX Control Kit V2 Illumina CT 901 2001 Components PhiX Control Table 40 NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S Component NEBNext RNA Fragmentation Buffer 10X NEBNext RNA Fragmentation Stop Solution 10X Linear Acrylamide 10 mg mL Random Primers 3 pg pL Murine RNase Inhibitor NEBNext First Strand Synthesis Reaction Buffer bX NEBNext Second Strand Synthesis Enzyme Mix NEBNext Second Strand Synthesis Reaction Buffer 10X Phosphorylation Reaction Buffer 10X Deoxynucleotide Solution Mix 10 mM each dNTP T4 DNA Polymerase DNA Polymerase Large Klenow Fragment T4 Polynucleotide Kinase Deoxyadenosine 5
7. Illumina Prepped library Production 2 days Library Hybridization 25 hours optional 72 hours Bead preparation 30 minutes Capture Selection and Washing 2 hours DNA purification 30 minutes Post Hybridization Amplification 1 hour PCR purification 30 minutes Bioanalyzer QC 1 hour SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 17 2 Sample Preparation Step 1 Purify the mRNA Purify 1 to 10 ug of total RNA Use the purification kit of your choice Step 2 Fragment the RNA Use reagents from the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E61008 NEBNext RNA Fragmentation Buffer 10X NEBNext RNA Fragmentation Stop Solution 10X 1 Preheat a PCR thermal cycler to 94 C 2 Add the components in Table 12 to a 200 uL thin wall PCR tube Table 12 Fragmentation Buffer Mix Component Volume NEBNext RNA Fragmentation Buffer 10X 2 uL Purified mRNA 1to 18 uL Nuclease free Water enough to bring total volume to 20 uL Total 20 uL Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 3 Incubate the tube in a preheated PCR thermal cycler at 94 C for exactly 5 minutes 4 Add 2 uL of NEBNext RNA Fragmentation Stop Solution 10X 5 Putthe tube on ice 18 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 3 Clean up the fragmented mRNA In this step you precipitate
8. Nuclease free 1 5 mL microfuge tubes sustainable Ambion p n AM12400 or equivalent at 95 C Thermal cycler Agilent SureCycler Life Technologies Veriti Thermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent Nuclease free 0 2 mL PCR tubes thin walled Eppendorf p n 951010006 or equivalent Microcentrifuge Eppendorf Microcentrifuge Model 5417C or equivalent P10 P20 P200 and P1000 pipettes Pipetman P10 P20 P200 P1000 or equivalent Vacuum concentrator Savant SpeedVac or equivalent Ice bucket Powder free gloves Sterile nuclease free aerosol barrier pipette tips Timer Vortex mixer Heat block at 37 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Before You Begin 1 Table 8 Required Equipment for Hybridization Description Vendor and part number Mx3000P Mx3005P 96 well tube plates Agilent p n 410088 or equivalent Mx3000P Mx3005P optical strip caps Agilent p n 401425 or equivalent MicroAmp Clear Adhesive Film Life Technologies p n 4306311 or equivalent BD Clay Adams Nutator Mixer BD Diagnostics p n 421105 or equivalent Dynal DynaMag 2 magnetic stand Life Technologies p n 123 21D or equivalent P10 P20 P200 and P1000 pipettes Pipetman P10 P20 P200 P1000 or equivalent Pipet Light Multichannel Pipette 12 channels Rainin p n L12 20 or equivalent Sterile nuclease free aerosol barrier pipette tips Thermal cycler Agilent SureCycler Life Technologies Veriti T
9. T Indexes for Illumina on page 73 1 For 1 library In a PCR tube strip tube or plate prepare the reaction mix in Table 27 on ice Mix well by gently pipetting up and down 2 For multiple libraries a Prepare the reaction mix in Table 27 on ice Mix well on a vortex mixer b Add 36 uL of the reaction mix to each well or tube c Add 1 uL of the appropriate index PCR Primer Index 1 through Index 16 clear caps from the SureSelect RNA Primer Kit to each well and mix by pipetting Use a different index primer for each sample to be sequenced in the same lane SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 4 Step 1 Amplify the sample to add index tags d Use a pipette to add 14 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples to avoid cross contamination Table 27 Herculase Il Master Mix Reagent Volume for 1 Volume for 12 reactions reaction includes excess Captured DNA 14 uL Nuclease free water 22 5 uL 281 25 uL 5X Herculase II Rxn Buffer clear cap 10 uL 125 pL 100 mM dNTP Mix green cap 0 5 pL 6 25 uL Herculase II Fusion DNA Polymerase red cap 1pyL 12 5 uL SureSelect ILM Indexing Post Capture Forward 1pyL 12 5 pL PCR Primer orange cap PCR Primer Index 1 through Index 16 clear caps 1 uL Included in the Herculase Il Fusion DNA Polymerase Agi
10. Triphosphate dATP 1 0 mM Klenow Fragment 3 5 exo NEBuffer 2 for Klenow Fragment 3 5 exo 10X Quick T4 DNA Ligase Quick Ligation Reaction Buffer 2X Nuclease free water SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 71 5 72 Reference Table 40 NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S continued Component PhusionR High Fidelity DNA Polymerase manufactured by Finnzymes Oy PhusionR HF Buffer bX manufactured by Finnzymes Oy Table 41 OlAquick PCR Purification Kit Qiagen p n 28104 Components OlAquick spin column Buffer PB Buffer PE concentrate Buffer EB pH Indicator collection tube 2 mL loading dye Contain chaotropic salts which are irritants Take appropriate laboratory safety measures and wear gloves when handling SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Reference 5 SureSelect T Indexes for Illumina The nucleotide sequence of each of the SureSelect index is listed in Table 42 Table 42 SureSelect T Indexes 1 16 Index Number Sequence 1 ATCACG 2 CGATGT 3 TTAGGC 4 TGACCA 5 ACAGTG 6 GCCAAT 7 CAGATC 8 ACTTGA 9 GATCAG 10 TAGCTT 11 GGCTAC 12 CTTGTA 13 AAACAT 14 CAAAAG 15 GAAACC 16 AAAGCA SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 73 5 74 Reference Alternative Cap
11. paired end adaptor 29 Step 12 Purify the sample with the QlAquick PCR Purification Kit 30 Step 13 Size select the DNA fragments with a E Gel SizeSelect 2 Agarose gel 31 Step 14 Purify the sample with the QlAquick PCR Purification Kit 33 Step 15 Amplify adaptor ligated library 34 Step 16 Purify the sample with the QlAquick PCR Purification Kit 37 Step 17 Assess quality and quantity with 2100 Bioanalyzer 38 This section contains instructions for prepped library production specific to the Illumina read sequencing platform It is intended for use with the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E61008 Refer to the NEBNext mRNA Sample Prep Reagent Set 1 Instruction Manual for more information E Agilent Technologies 15 2 Sample Preparation Total RNA or mRNA ss Purify the mRNA m RT 2nd strand synthesis Double s ded cDNA End repair MTT EL LE saa Phosphorylated ends Y Add Klenow and dATP 3 dA overhang Ligate adaptors ranscripts Bait Design in eArray ii ibra ridization 24 hours at 65 C ybri apture election Magnetic bead selection PCR and purification Uail ssessmen Bioanalyzer and Quantitation Se uencin Figure 1 Overall sequencing sample preparation workflow 16 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Table 11 Overview and time requirements Step Time
12. sample using Agencourt AMPure XP beads 53 4 Addition of Index Tags by Post Hybridization Amplification 55 5 Step 1 Amplify the sample to add index tags 56 Step 2 Purify the sample using Agencourt AMPure XP beads 59 Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNAassay 60 Step 4 Assess the quantity of each index tagged library by OPCR 62 Step 5 Pool samples for Multiplexed Sequencing 63 Step 6 Prepare sample for cluster amplification 65 Reference 67 SureSelect Reagent Kit Content 68 Other Reagent Kits Content 70 SureSelect T Indexes for Illumina 73 Alternative Capture Equipment Combinations 74 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Protocol ee 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 12 Optional Equipment 14 Make sure you have the most current protocol Go to the SureSelect Related Literature page on genomics agilent com and search for manual number G7580 90010 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non Agilent protocols to process samples for enrichment E Agilent Techn
13. the beads at this time Stopping Point If you do not continue to the next step store the samples at 4 C for up to a week or at 20 C for longer periods SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 59 4 Addition of Index Tags by Post Hybridization Amplification Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay As an alternative you can use the High Sensitivity D1K ScreenTape Agilent p n 5067 5363 and High Sensitivity D1K Reagents Agilent p n 5067 5364 For more information to do this step see the Agilent 2200 TapeStation User Manual Use a Bioanalyzer High Sensitivity DNA Assay to assess the quality and size range Note that the concentration of each sample loaded on the chip must be within the linear range of the assay to accurately quantify 5 pg to 500 pg You may need to dilute your sample accordingly Refer to the Agilent High Sensitivity DNA Kit Guide at http www chem agilent com en US Search Library _layouts Agilent PublicationSummary aspx whid 59504 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100 Expert Software version B 02 07 or higher required to run the High Sensitivity Kit turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanaly
14. 13 000 rpm 12 Collect the eluate If you do not continue to the next step store the samples at 20 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 11 Ligate the paired end adaptor Use the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S and the SureSelect RNA Primer Kit 1 For 1 library prepare on ice In a PCR tube strip tube or plate prepare the reaction mix in Table 20 2 For multiple libraries prepare on ice a Prepare the reaction mix in Table 20 b Add 27 uL of the reaction mix to each well or tube c Add 23 uL of each cDNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 20 Ligation master mix Reagent Volume for 1 Library Volume for 12 Libraries includes excess Purified dA Tailed DNA sample 23 uL Quick Ligation Reaction Buffer 2X 25 uL 312 5 pL Quick T4 DNA Ligase 1 0 pL 12 5 pL SureSelect Adaptor Oligo Mix brown cap 1 0 uL 12 5 uL Total Volume 50 pL 337 5 pL 27 pL sample Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S t Included in the SureSelect RNA Primer Kit 3 Incubate for 15 minutes at 20 C on a thermal cycler Do not use a heated lid SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 29 2 Sample Preparation Stopping Point 30 Step 12 Purify the sample with the OlAquic
15. 5190 4456 5190 4457 Indexing Hyb Module Box 2 SureSelect RNA Primer Kit 20 C 5190 5337 5500 5338 The content of each of these kits are described in the next tables Table 32 SureSelect Target Enrichment Kit Box 1 Kit Component SureSelect Hyb 1 orange cap or bottle SureSelect Hyb 2 red cap SureSelect Hyb 4 black cap or bottle SureSelect Binding Buffer SureSelect Wash 1 SureSelect Wash 2 SureSelect Elution Buffer SureSelect Neutralization Buffer SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Reference 5 Table 33 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Kit Component SureSelect Hyb 3 yellow cap SureSelect Indexing Block 1 green cap SureSelect Block 2 blue cap SureSelect Indexing Block 3 brown cap SureSelect RNase Block purple cap SureSelect ILM Indexing Pre Capture PCR Reverse Primer clear cap SureSelect ILM Indexing Post Capture Forward PCR Primer orange cap Table 34 SureSelect RNA Primer Kit Kit Component SureSelect Adaptor Oligo Mix brown cap SureSelect Primer brown cap PCR Primer Index 1 through Index 16 clear caps SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 69 5 70 Other Reagent Kits Content These reagents are from kits other than the SureSelect Reagent kit Make sure you use only the reagents listed here Table 35 Herculase
16. 80 uL of sample and mix well by pipetting Check for the yellow color to make sure Buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen QIAquick Handbook If needed use 5 uL of the 3M Sodium Acetate included in the kit to adjust the pH to the proper range 4 Puta QIAquick spin column in a 2 mL collection tube 5 Transfer the 480 uL sample to the QIAquick spin column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 6 Add 750 uL of Buffer PE concentrate to the column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 7 Putthe QIAquick spin column back in the collection tube 2 mL and spin in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm 8 Transfer the QIAquick spin column to a new 1 5 mL microcentrifuge tube to elute the cleaned sample 9 Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 50 uL of Buffer EB directly onto the QIAquick filter membrane 11 Wait 60 seconds then centrifuge for 60 seconds at 17 900 x g 13 000 rpm 12 Collect the eluate Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 23 2 24 Sample Preparation Step 7 Repair the ends To process multiple samples prepare master mixe
17. Expert Software version B 02 02 or higher turn on the 2100 Bioanalyzer and check communication 3 Prepare the chip samples and ladder as instructed in the reagent kit guide 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Check that the electropherogram shows a distribution with a peak size approximately 200 to 250 bp Measure the concentration of the library by integrating under the peak 38 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 17 Assess quality and quantity with 2100 Bioanalyzer Fu Sample 7 1 SO 100 150 20 300 o0 so 700 1500 te Figure 3 Analysis of amplified prepped library DNA using a DNA 1000 assay The elec tropherogram shows a single peak in the size range of 200 to 250 bp SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 39 2 Sample Preparation Step 17 Assess quality and quantity with 2100 Bioanalyzer 40 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing CAUTION SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Protocol ece 3 Hybridization Step 1 Hybridize the library 44 Step 2 P
18. Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol 8 Repeat step 6 and step 7 step once 9 Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol completely evaporates Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 30 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind tube You can discard the beads at this time Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 53 3 Hybridization Step 4 Purify the sample using Agencourt AMPure XP beads 54 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Protocol 4 Addition of Index Tags by Post Hybridization Amplification Step 1 Amplify the sample to add index tags 56 Step 2 Purify the sample using Agencourt AMPure XP beads 59 Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 60 Step 4 Assess the quantity of each index tagg
19. Step 17 Assess quality and quantity with 2100 Bioanalyzer on page 38 After quantitation adjust the sample to 30 ng uL Alternatively concentrate a 100 ng aliquot at 45 C down to 3 4 uL If the sample dries up completely resuspend in 3 4 uL of water and mix on a vortex mixer If processing multiple samples adjust to equivalent volumes before concentrating 3 Mix the components in Table 24 at room temperature to prepare the hybridization buffer 44 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Hybridization 3 Table 24 Hybridization Buffer Reagent Volume for 1 Volume for 6 Volume for 12 capture pL captures pL captures pL includes excess includesexcess includes excess SureSelect Hyb 1 orange cap or 25 125 250 bottle SureSelect Hyb 2 red cap 1 5 10 SureSelect Hyb 3 yellow cap 10 50 100 SureSelect Hyb 4 black cap or 13 65 130 bottle Total 49 40 pL 245 40 490 40 uL sample needed uL sample 4 If precipitate forms warm the hybridization buffer at 65 C for 5 minutes 5 In a PCR plate strip tubes or tubes prepare the SureSelect capture library mix for target enrichment a Keep tubes on ice until step 10 b For each sample add 5 uL of SureSelect capture library c For 1 library combine 1 uL SureSelect RNase Block purple cap with 2 uL nuclease free water For multiple libraries use 1 part SureSelect RNase Block purple cap to Z parts nuclea
20. SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Protocol Version 2 2 1 February 2012 SureSelect platform manufactured with Agilent SurePrint Technology Research Use Only Not for use in Diagnostic Procedures RE Agilent Technologies Notices Agilent Technologies Inc 2011 2012 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number G7580 90010 Edition Version 2 2 1 February 2012 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Rd Santa Clara CA 95051 USA Acknowledgement Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Notice to Purchaser Research Use Only Not for use in diagnostic procedures Warranty The material contained in this document is provided as is
21. ated lid 8 Add 1 uL of SuperScript II Reverse Transcriptase to the sample 9 Run the program listed in Table 16 in a thermal cycler to incubate the sample Use a heated lid at 105 C Table 16 PCR program Temperature Time 25 C 10 minutes 42 C 50 minutes 70 C 15 minutes 4 C Hold 10 Put the tube on ice SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 21 2 22 Sample Preparation Step 5 Synthesize second strand cDNA Use reagents from NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 1 Preheat a PCR thermal cycler to 16 C 2 Add 48 uL of Nuclease Free Water to the first strand cDNA synthesis mix 3 Add the reagents the in Table 17 to the mix Table 17 Component Volume NEBNext Second Strand Synthesis Reaction Buffer 10X 8 uL NEBNext Second Strand Synthesis Enzyme Mix 4 uL Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 4 Mix well by gentle pipetting 5 Incubate the sample at 16 C for 2 5 hours in a thermal cycler Use a heated lid at 50 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 6 Purify the sample with the OlAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit Qiagen p n 28104 1 If you haven t already done so add the pH Indicator I to the Buffer PB 2 Add 400 uL of Buffer PB to
22. cluded in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S except where indicated t Included in the SureSelect RNA Primer Kit 3 Run the program listed in Table 23 in a thermal cycler to amplify the sample Use a heated lid at 105 C 4 Amplify using the following PCR program SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 35 2 36 Sample Preparation Table 23 PCR program Step Temperature Time Step 1 98 C 30 seconds Step 2 98 C 10 seconds Step 3 65 C 30 seconds Step 4 72 C 30 seconds Step 5 Repeat Step 2 through Step 4 for a total of 15 times Step 6 72 C 5 minutes Step 7 4 C Hold SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 16 Purify the sample with the OlAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit Qiagen p n 28104 1 If you haven t already done so add the pH Indicator I to the Buffer PB 2 Add 250 uL of Buffer PB to 50 uL of sample and mix well by pipetting Check for the yellow color to make sure Buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen QIAquick Handbook If needed use 5 uL of the 3M Sodium Acetate included in the kit to adjust the pH to the proper range 4 Put a QIAquick spin column in a 2 mL collection tube 5 Transfer the 300 uL of sample to the QIAquick spin colum
23. ct RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 19 2 20 Sample Preparation Step 4 Synthesize first strand cDNA Use reagents from the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S and the SuperScript II Reverse Transcriptase 1 Add the components in Table 14 to a 200 uL thin wall PCR tube Table 14 Random Primer Mix Component Volume Random Primers 3 ug L 1 uL mRNA 13 5 pL Total 14 5 uL Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 2 Incubate the sample in a PCR thermal cycler at 65 C Use a heated lid at 105 C 3 Spin the sample briefly and then put the tube on ice 4 Setthe PCR thermal cycler to 25 C Mix the reagents in Table 15 in the order listed in a separate tube Prepare 10 extra reagent mix if you are preparing multiple samples Table 15 Random Primer Mix Component Volume for 1 Volume for 10 reaction reactions with excess NEBNext First Strand Synthesis Reaction Buffer 5X 4 uL 44 uL Murine RNase Inhibitor 0 5 uL 5 5 uL Total 4 5 uL 49 5 uL Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 6 Add 4 5 uL of mixture to the PCR tube and mix well SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 7 Heat the sample in the preheated PCR thermal cycler at 25 C for 2 minutes Do not use a he
24. e or tubes at 65 C in the PCR machine while you add the hybridization mixture directly from the thermal cycler to the bead solution Invert the tube to mix 3 to 5 times Excessive evaporation such as when less than 20 uL remains after hybridization can indicate suboptimal capture performance See Table 43 on page 74 for tips to minimize evaporation 3 Incubate the hybrid capture bead solution on a Nutator or equivalent for 30 minutes at room temperature Make sure the sample is properly mixing in the tube 4 Briefly spin in a centrifuge Separate the beads and buffer on a magnetic separator and remove the supernatant 6 Resuspend the beads in 500 uL of SureSelect Wash 1 by mixing on a vortex mixer for 5 seconds 7 Incubate the samples for 15 minutes at room temperature 8 Separate the beads and buffer on a magnetic separator and remove the supernatant SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 51 3 52 Hybridization 9 Wash the beads a Resuspend the beads in 500 uL of 65 C prewarmed SureSelect Wash 2 and mix on a vortex mixer for 5 seconds to resuspend the beads b Incubate the samples for 10 minutes at 65 C in a recirculating water bath heat block or equivalent Do not use a tissue incubator It cannot properly maintain temperature c Invert the tube to mix The beads may have settled d Separate the beads and buffer on a magnetic separator and remove the
25. earch PTC 200 410088 Mx3000 3005 401425 Mx3000 3005 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing www agilent com In This Book This guide contains information to run the SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing protocol O Agilent Technologies Inc 2011 2012 Version 2 2 1 February 2012 G7580 90010 Revision B2 OE Agilent Technologies
26. ed library by OPCR 62 Step 5 Pool samples for Multiplexed Sequencing 63 Step 6 Prepare sample for cluster amplification 65 This chapter describes the steps to add index tags by amplification purify and assess quality and quantity of the libraries and dilute the sample appropriately for cluster amplification and pool indexed samples for multiplexed sequencing XE Agilent Technologies 55 4 Addition of Index Tags by Post Hybridization Amplification CAUTION Step 1 Amplify the sample to add index tags Use reagents from Herculase II Fusion DNA Polymerase Agilent SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 SureSelect RNA Primer Kit Do not use amplification enzymes other than Herculase Il Fusion DNA Polymerase Other enzymes have not been validated CAUTION This protocol was optimized to minimize PCR based bias in the library preparation To determine the number of cycles needed do a trial amplification with 12 cycles If you do not get enough yield for Illumina sequencing repeat with 14 cycles CAUTION To avoid cross contaminating libraries set up PCR reactions all components except the library DNA in a dedicated clean area or PCR hood with UV sterilization and positive air flow 56 Prepare 1 amplification reaction for each hybrid capture Include a negative no template control To see the nucleotide sequence in each of the index included in SureSelect reagent kits see SureSelect
27. ent p n 5067 4626 Herculase II Fusion DNA Polymerase Agilent includes dNTP mix and 5x Buffer 200 reactions p n 600677 400 reactions p n 600679 Nuclease free Water not DEPC treated Ambion Cat AM9930 1X Low TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA Life Technologies p n 4389764 E Gel SizeSelect 2 Agarose Gel Life Technologies p n G6610 02 SuperScript Il Reverse Transcriptase Life Technologies 2 000 units p n 18064 022 10 000 units p n 18064 014 4x10 000 units p n 18064 071 NEBNext mRNA Sample Prep Reagent Set 1 New England BioLabs p n E6100S Buffer EB 10mM Tris Cl ph 8 5 Qiagen p n 19086 100 Ethanol molecular biology grade Sigma Aldrich p n E7023 3 M NaOAc pH 5 2 Distilled water SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 9 1 Before You Begin Table 2 Required Reagents for Cluster Generation and Sequencing Description Vendor and part number Illumina Cluster Generation Kit depending on your instrument and setup TruSeq PE Cluster Kit vb CS GA TruSeq PE Cluster Kit v2 cBot HS TruSeq PE Cluster Kit v2 5 cBot HS PhiX Control Kit V2 for HiSeq 2000 Illumina Sequencing Kit depending on your instrument and setup TruSeq SBS Kit v5 GA 36 cycle TruSeq SBS Kit HS 50 cycle Illumina p n PE 203 5001 Illumina p n PE 401 2001 Illumina p n PE 401 2510 Illumina p n CT 901 2001 Illumina p n FC 104 5001 Illumina p n FC 401 1002 Table3 SureSelect Reagent Kit
28. es Step 2 65 C Hold 8 Use a heated lid on the thermal cycler at 105 C to hold the temperature of the plate on the thermal cycler at 65 C CAUTION The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 9 Maintain the plate at 65 C while you load 40 uL of hybridization buffer per well into the A row of the PCR plate The number of wells filled in Row A is the number of libraries prepared The example in Figure 6 is for 12 captures Hybridization Buffer Prepped Library Figure 6 Hybridization buffer shown in blue Make sure that the plate is at 65 C for a minimum of 5 minutes before you go to step 10 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 47 3 Hybridization 10 Add the capture library mix from step 5 to the PCR plate a Add the capture library mix 7 uL to the C row in the PCR plate For multiple samples use a multi channel pipette to load the capture library mix into the C row in the PCR plate Keep the plate at 65 C during this time b Seal the wells with strip caps Use a capping tool to make sure the fit is tight c Incubate the samples at 65 C for 2 minutes 11 Maintain the plate at 65 C while you use a multi channel pipette to take 13 uL of Hybridization Buffer from the A row and add it to the SureSelect capture library mix contained in row C of the PCR plate for each sample See Figu
29. exed Sequencing 61 4 4 Addition of Index Tags by Post Hybridization Amplification Step 4 Assess the quantity of each index tagged library by OPCR Refer to the protocol that is included with the QPCR NGS Library Quantification Kit Illumina GA for more details to do this step 1 Use the QPCR NGS Library Quantification Kit Illumina GA to determine the concentration of each index tagged captured library 2 Prepare a standard curve using the quantification standard included in the kit according to the instructions provided in the user guide 3 Dilute each index tagged captured library such that it falls within the range of the standard curve Typically this corresponds to approximately a 1 1000 to 1 10 000 dilution of the captured DNA 4 Prepare the QPCR master mix with Illumina adaptor specific PCR primers according to instructions provided in the kit 5 Add an aliquot of the master mix to PCR tubes and add template 6 On a QPCR system such as the MX3005P run the thermal profile outlined in the QPCR NGS Library Quantification kit user guide Use the SYBR Green instrument setting 7 Usethe standard curve to determine the concentration of each unknown index tagged library in nM The concentration will be used to accurately pool samples for multiplexed sequencing 62 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 4 Step 5 Po
30. f Buffer EB directly onto the QIAquick filter membrane 11 Wait 60 seconds then centrifuge for 60 seconds at 17 900 x g 13 000 rpm 12 Collect the eluate 26 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 9 Add dA Bases to the 3 end of the cDNA fragments Use the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100 1 For 1 library prepare on ice In a PCR tube strip tube or plate prepare the reaction mix in Table 19 Mix well by gently pipetting up and down 2 For multiple libraries prepare on ice a Prepare the reaction mix in Table 19 Mix well on a vortex mixer b Add 18 uL of the reaction mix to each well or tube c Add 32 uL of each DNA sample to each well or tube Mix by pipetting Change pipette tips between samples Table 19 Adding A Bases Reagent Volume for 1 Library Volume for 12 Libraries includes excess Purified end repaired cDNA sample 32 uL NEBuffer 2 for Klenow Fragment 3 5 exo 10X 5 pL 62 5 uL Deoxyadenosine 5 Triphosphate dATP 1 0 mM 10 uL 125 uL Klenow Fragment 3 5 exo 3 uL 37 5 uL Total Volume 50 uL 225 uL 18 pL sample Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 3 Incubate in a thermal cycler for 30 minutes at 37 C Do not use a heated lid SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Se
31. he NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 2 25 2 Sample Preparation Step 8 Purify the sample with the OlAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit Qiagen p n 28104 1 If you haven t already done so add the pH Indicator I to the Buffer PB 2 Add 500 uL of Buffer PB to 100 uL of sample and mix well by pipetting Check for the yellow color to make sure Buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen QIAquick Handbook If needed use 5 uL of the 3M Sodium Acetate included in the kit to adjust the pH to the proper range 4 Puta QIAquick spin column in a 2 mL collection tube 5 Transfer the 600 uL of sample to the QIAquick spin column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 6 Add 750 uL of Buffer PE concentrate to the column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 7 Putthe QIAquick spin column back in the collection tube 2 mL and spin in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm 8 Transfer the QIAquick spin column to a new 1 5 mL microcentrifuge tube to elute the cleaned sample 9 Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 32 uL o
32. he sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 90 uL of homogenous AMPure beads to a 1 5 mL LoBind tube and add sample library 50 uL Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 500 uL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result 7 Let the tube sit for 1 minute to allow any disturbed beads to settle and remove the ethanol 8 Repeat step 6 and step 7 once 9 Dry the samples on the 37 C heat block for 5 minutes or until the residual ethanol is completely evaporated Do not dry the bead pellet to the point that the bead pellet appears cracked Elution efficiency is significantly decreased when the bead pellet is excessively dried 10 Add 30 uL nuclease free water mix well on a vortex mixer and incubate for 2 minutes at room temperature 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes until the solution is clear 12 Remove the supernatant 30 uL to a fresh 1 5 mL LoBind tube You can discard
33. hermal Cycler BioRad MJ Research DNA Engine PTC 200 or equivalent Timer Vortex mixer Water bath or heat block set to 65 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 13 1 14 Before You Begin Optional Equipment Table 9 Optional Equipment for Hybridization Description Vendor and part number Tube strip capping tool Agilent p n 410099 Table 10 Optional Equipment for Library Prep and Post Hybridization Amplification Description Vendor and part number 2200 TapeStation System Agilent p n G2964AA or G2965AA D1K ScreenTape Agilent p n 5067 5361 D1K Reagents Agilent p n 5067 5362 High Sensitivity D1K ScreenTape Agilent p n 5067 5363 High Sensitivity D1K Reagents Agilent p n 5067 5364 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Protocol 2 Sample Preparation Step 1 Purify the mRNA 18 Step 2 Fragment the RNA 18 Step 3 Clean up the fragmented mRNA 19 Step 4 Synthesize first strand cDNA 20 Step 5 Synthesize second strand cDNA 22 Step 6 Purify the sample with the QlAquick PCR Purification Kit 23 Step 7 Repair the ends 24 Step 8 Purify the sample with the OlAquick PCR Purification Kit 26 Step 9 Add dA Bases to the 3 end of the cDNA fragments 27 Step 10 Purify the sample with the QlAquick PCR Purification Kit 28 Step 11 Ligate the
34. inal dilution Dilute 8 uL of denatured DNA with 992 uL of pre chilled HT1 Hybridization Buffer for a final concentration of 12 pM ao a FF N Mix the sample briefly on a vortex mixer and pulse centrifuge 8 Continue with cluster generation Use the TruSeq SBS Kit v5 GA 36 cycle and the appropriate Illumina multiplexed sequencing protocol SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 65 4 66 Addition of Index Tags by Post Hybridization Amplification HiSeq2000 with PhiX spike in controls Use reagents from the appropriate for your instrument HT1 Hybridization Buffer HP3 2 N NaOH Use the PhiX Control Kit V2 lumina CT 901 2001 for PhiX Control Prepare a 1 20 dilution of HP3 2 N NaOH down to 0 1N NaOH 2 Prepare 10 nM 10 fmol uL dilutions of the amplified capture based on the Bioanalyzer quantitation 3 Add 20 fmol 2 uL of the 10 nM multiplexed sample pool into 8 uL of Buffer EB 10mM Tris Cl ph 8 5 to make a 2 nM solution Add 10 uL of 0 1 N NaOH Mix the sample briefly on a vortex mixer and pulse centrifuge Incubate for 5 minutes at room temperature to denature the DNA Add 980 uL of HT1 Hybridization Buffer to the denatured DNA to make 20 pM template solution uy oO GC A co Mix the sample briefly on a vortex mixer and pulse centrifuge 9 Prepare 4 pM template by mixing 200 uL of 20 pM solution with 800 uL of Pre Chilled HT1 Hybridization Buffe
35. k PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit Qiagen p n 28104 If you haven t already done so add the pH Indicator I to the Buffer PB 2 Add 250 uL of Buffer PB to 50 uL of sample and mix well by pipetting Check for the yellow color to make sure Buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen QIAquick Handbook If needed use 5 uL of the 3M Sodium Acetate included in the kit to adjust the pH to the proper range 4 Puta QIAquick spin column in a 2 mL collection tube 5 Transfer the 300 uL of sample to the QIAquick spin column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through Add 750 uL of Buffer PE concentrate to the column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through Put the QIAquick spin column back in the collection tube 2 mL and spin in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Transfer the QIAquick spin column to a new 1 5 mL microcentrifuge tube to elute the cleaned sample Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 10 uL of Buffer EB directly onto the QIAquick filter membrane 11 Wait 60 seconds then centrifuge for 60 seconds at 17 900 x g 13 000 rpm 12 Collect the eluate If you do not continue to the next step store the samples at 20
36. lent Do not use the buffer or dNTP mix from any other kit t Included in the SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Use one of the 16 primers included in the SureSelect RNA Primer Kit 3 Putthe samples in a thermal cycler with a heated lid at 105 C Run the program listed in Table 28 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 57 4 Addition of Index Tags by Post Hybridization Amplification Table 28 PCR program Step Temperature Time Step 1 98 C 30 seconds Step 2 98 C 10 seconds Step 3 57 C 30 seconds Step 4 72 C 30 seconds Step 5 Repeat Step 2 through Step 4 for a total of 12 to 16 times Step 6 72 C 5 minutes Step 7 4 C Hold As with the pre capture PCR amplification minimize the number of PCR cycles used to enrich the captured DNA The use of only half of the captured DNA for amplification lets you adjust the number of cycles by repeating the PCR if needed Use the optimal cycle number to repeat PCR at the 50 uL reaction scale See Table 29 for approximate number of cycles for a given library size Results may vary based on library content Table 29 Cycle times Capture Size Cycles 1 kb up to 0 5 Mb 16 cycles 0 5 Mb up to 1 49 Mb 14 cycles gt 1 5 Mb 12 cycles 58 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 4 Step 2 Purify t
37. n Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 6 Add 750 uL of Buffer PE concentrate to the column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 7 Putthe QIAquick spin column back in the collection tube 2 mL and spin in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm 8 Transfer the QIAquick spin column to a new 1 5 mL microcentrifuge tube to elute the cleaned sample 9 Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 30 uL of Buffer EB directly onto the QIAquick filter membrane 11 Wait 60 seconds then centrifuge for 60 seconds at 17 900 x g 13 000 rpm 12 Collect the eluate Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 37 2 Sample Preparation Step 17 Assess quality and quantity with 2100 Bioanalyzer As an alternative you can use the D1K ScreenTape Agilent p n 5067 5361 and D1K Reagents Agilent p n 5067 5362 For more information to do this step see the Agilent 2200 TapeStation User Manual Use the Bioanalyzer DNA 1000 to assess the quantity quality and size distribution of the PCR products 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide 2 Open the Agilent 2100
38. ol samples for Multiplexed Sequencing 1 Combine the libraries such that each index tagged sample is present in equimolar amounts in the pool For each library use the formula below to determine the amount of index sample to use Volume of Index VO x CG where x C i V f is the final desired volume of the pool C f is the desired final concentration of all the DNA in the pool for example 10 nM for the standard Illumina protocol is the number of index and Ci is the initial concentration of each index sample Table 30 shows an example of the amount of 4 index tagged of different concentrations and Low TE needed for a final volume of 20 uL at 10 nM Table 30 Example of index volume calculation for a total volume of 20 uL Component V f C i C f Volume to use pL Sample 1 20 pL 20 nM 10 nM 4 2 5 Sample 2 20 pL 10 nM 10 nM 4 5 Sample 3 20 pL 17 nM 10 nM 4 2 9 Sample 4 20 uL 25 nM 10 nM 4 2 Low TE 7 6 2 Adjust the final volume of the pooled library to the desired final concentration If the final volume of the combined index tagged samples is less than the desired final volume V f add Low TE to bring the volume to the desired level If the final volume of the combined index tagged samples is greater than the final desired volume V f lyophilize and reconstitute to the desired volume 3 If you store the library before sequencing add Tween 20 to 0 1 v v and store at 20 C short term S
39. ologies 1 Before You Begin Procedural Notes Prolonged exposure of SureSelect Elution Buffer to air can decrease product performance by altering the pH of the solution Keep the Elution Buffer container tightly sealed when not in use To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips Maintain a clean work area Do not mix stock solutions and reactions containing RNA on a vortex mixer Instead gently tap the tube with your finger to mix the sample Avoid repeated freeze thaw cycles of stock and diluted RNA solutions When preparing frozen reagent stock solutions for use 1 Thaw the aliquot as rapidly as possible without heating above room temperature 2 Mix briefly on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid 3 Store on ice or in a cold block until use In general follow Biosafety Level 1 BL1 safety rules Safety Notes CAUTION Wear appropriate personal protective equipment PPE when working in the laboratory 8 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Before You Begin 1 Required Reagents Table 1 Required Reagents for Library Prep and Post Hybridization Amplification Description Vendor and part number DNA 1000 Kit Agilent p n 5067 1504 High Sensitivity DNA Kit Agil
40. ore you start an experiment 2 Sample Preparation This chapter describes the steps to prepare the RNA sample for target enrichment 3 Hybridization This chapter describes the steps to prepare and hybridize samples 4 Addition of Index Tags by Post Hybridization Amplification This chapter describes the steps to amplify purify and assess quality of the sample library SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 3 What s New in 2 2 What s New in 2 1 What s New in 2 0 What s New in 1 1 New product configuration and product numbers for SureSelect reagent kits and capture libraries Support for the optional use of the Agilent 2200 TapeStation for RNA quantitation and qualification Support for custom SureSelect RNA capture libraries SureSelect RNA Primer Kit for Illumina replaces SureSelect Library Prep Kit Dynabeads MyOne Streptavidin T1 beads replaces Agilent LodeStars 2 7 Streptavidin beads Agilent LodeStars 2 7 Streptavidin beads replaces Dynabeads MyOne Streptavidin T1 beads NEBNext mRNA Sample Prep Reagent Set 1 replaces the Ilumina mRNA Seq Prep Kit QIAquick PCR Purification Kit replaces Agencourt AMPure XP beads for RNA purification Agarose gel purification step is added in sample preparation Reagent cap colors are listed where available More details given for the reagent kits to use for each step Update to cluster generation reagents and procedure
41. protocol was optimized to minimize PCR based bias in the library preparation While most library preparations yield enough cDNA 100 ng for at least a single hybridization poor quality RNA samples or other factors can affect yield 34 Use reagents in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S the SureSelect RNA Primer Kit 1 For 1 library prepare on ice In a PCR tube strip tube or plate prepare the reaction mix in Table 22 Mix well by gently pipetting up and down 2 For multiple libraries prepare on ice a Prepare the reaction mix in Table 22 Mix well on a vortex mixer b Add 21 uL of the reaction mix to each well or tube c Add 29 uL of each cDNA sample to each well or tube Mix by pipetting Change pipette tips between samples SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Sample Preparation 2 Step 15 Amplify adaptor ligated library Table 22 PCR Components Reagent Volume for 1 Volume for 12 Libraries Library Size selected cDNA 29 uL PhusionR HF Buffer 5X manufactured by 10 pL 125 uL Finnzymes Oy SureSelect Primer brown cap Tul 12 5 pL SureSelect ILM Indexing Pre Capture PCR 1yL 12 5 pL Reverse Primer clear cap Deoxynucleotide Solution Mix 10 mM each 1 5 pL 18 75 uL dNTP Nuclease Free Water 7 uL 87 5 uL PhusionR High Fidelity DNA Polymerase 0 5 uL 6 25 uL manufactured by Finnzymes Oy In
42. quencing 27 2 Sample Preparation Stopping Point 28 Step 10 Purify the sample with the OlAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit Qiagen p n 28104 If you haven t already done so add the pH Indicator I to the Buffer PB 2 Add 250 uL of Buffer PB to 50 uL of sample and mix well by pipetting Check for the yellow color to make sure Buffer PB pH is correct For more information on how to check buffer pH refer to the Qiagen QIAquick Handbook If needed use 5 uL of the 3M Sodium Acetate included in the kit to adjust the pH to the proper range 4 Puta QIAquick spin column in a 2 mL collection tube 5 Transfer the 300 uL of sample to the QIAquick spin column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through Add 750 uL of Buffer PE concentrate to the column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through Put the QIAquick spin column back in the collection tube 2 mL and spin in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Transfer the QIAquick spin column to a new 1 5 mL microcentrifuge tube to elute the cleaned sample Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 23 uL of Buffer EB directly onto the QIAquick filter membrane 11 Wait 60 seconds then centrifuge for 60 seconds at 17 900 x g
43. r If densities higher than 400K 600K clusters mm are desired prepare a more concentrated sample from the 20 pM solution 10 Mix the sample briefly on a vortex mixer and pulse centrifuge 11 Remove 10 uL from solution 1000 uL to get 990 uL 12 Add 10 uL of PhiX Control 13 Mix the sample briefly on a vortex mixer and pulse centrifuge 14 Dispense 120 uL of diluted denatured sample DNA template and PhiX Control into a strip tube 15 Place on ice until ready to use 16 Continue with cluster generation Use TruSeq SBS Kit HS 50 cycle and the appropriate Illumina multiplexed sequencing protocol SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing s SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed e Sequencing Protocol 000 0905 5 659 Reference o o E e e i e j SureSelect Reagent Kit Content 68 e Other Reagent Kits Content 70 SureSelect T Indexes for Illumina 73 Alternative Capture Equipment Combinations 74 This chapter contains reference information ORE Agilent Technologies 67 5 Reference SureSelect Reagent Kit Content Each SureSelect Reagent Kit contains one or more of each of these individual kits Table 31 SureSelect Reagent Kit Contents Product Storage 16 96 480 Condition Reactions Reactions Reactions SureSelect Target Enrichment Kit Room 5190 4393 5190 4394 5190 4395 Box 1 Temperature SureSelect Target Enrichment Kit ILM 20 C 5190 4455
44. re 7 WA step 11 Hybridization Buffer Prepped Library SureSelect Capture Library Figure 7 SureSelect Capture Library or Baits shown in Green 12 Maintain the plate at 65 C while you use a multi channel pipette to transfer the entire contents of each prepped library mix in row B to the hybridization solution in row C See Figure 7 Mix well by slowly pipetting up and down 8 to 10 times The hybridization mixture is now 27 to 29 uL depending on degree of evaporation during the preincubations 13 Seal the wells with strip caps or double adhesive film Make sure all wells are completely sealed Use new adhesive seals or strip caps The structural integrity of the seals and caps can be compromised during the previous incubation steps 48 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Hybridization 3 CAUTION Wells must be adequately sealed to minimize evaporation or your results can be negatively impacted If you use strip tubes test for evaporation before you do the first experiment to make sure the tube capping method is appropriate for the thermal cycler Check that no more than 3 to 4 uL is lost to evaporation 14 Incubate the hybridization mixture for 24 hours at 65 C with a heated lid at 105 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 49 3 50 Hybridization Step 2 Prepare magnetic beads Use these reagent
45. repare magnetic beads 50 Step 3 Select hybrid capture with SureSelect 51 Step 4 Purify the sample using Agencourt AMPure XP beads 53 This chapter describes the steps to combine the prepped library with the hybridization reagents blocking agents and the SureSelect capture library The ratio of SureSelect capture library to prepped library is critical for successful capture E Agilent Technologies 41 3 Hybridization TOTAL RNA OR mRNA SureSelect WIV Target Enrichment System V0 A Capture Process J NGS Kit ERD emt AN cDNA LIBRARY PREPPED SureSelect HYB BUFFER BIOTINYLATED RNA LIBRARY BAITS Q O Hybridization 00000 9000 00000 STREPTAVIDIN COATED MAGNETIC BEADS 90006 27 00000 0006 MM LGR UNBOUND FRACTION Wash Beads DISCARDED and Digest RNA J Amplify VV NY Figure 4 SureSelect RNA Capture Process 42 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Hybridization 3 Refer to SureSelect Reagent Kit Content on page 68 for a complete content listing of each SureSelect RNA Capture kit CAUTION You must avoid evaporation from the small volumes of the capture during the 24 hour or greater incubation If you want to use a different combination of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape first test the conditions Incubate 27 uL of SureSelect Hybridization Buffer without DNA at 65 C for 24 hour
46. s or longer if applicable as a test Include buffer in each well that you might use including those in the center and those on the edges Check that you do not get extensive evaporation Evaporation should not exceed 3 to 4 pL For a partial list of tested options showing minimal evaporation refer to Alternative Capture Equipment Combinations on page 74 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 43 3 Hybridization Step 1 Hybridize the library The hybridization reaction requires 100 ng of cDNA with a maximum volume of 3 4 uL 1 Ifthe prepped library concentration is below 30 ng uL use a vacuum concentrator to concentrate the sample at lt 45 C a Addthe entire volume of prepped library to an Eppendorf tube Poke one or more holes in the lid with a narrow gauge needle You can also break off the cap cover with parafilm and poke a hole in the parafilm b Completely lyophilize Use a vacuum concentrator on low heat less than 45 C to dehydrate c Reconstitute with nuclease free water to bring the final concentration to 30 ng uL or greater if sample recovery is of concern Pipette up and down along the sides of the tube for optimal recovery d Mix well on a vortex mixer and spin in a microfuge for 1 minute 2 Optional To test recovery after lyophilization reconstitute the sample to greater than 30 ng uL and check the concentration on a Bioanalyzer DNA 1000 chip See
47. s from the SureSelect Target Enrichment Kit Box 1 SureSelect Binding Buffer SureSelect Wash 2 1 Prewarm SureSelect Wash 2 at 65 C in a circulating water bath for use in Step 3 Select hybrid capture with SureSelect 2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 on a vortex mixer Magnetic beads settle during storage 3 For each hybridization add 50 uL of Dynabeads MyOne Streptavidin T1 to a 1 5 mL microfuge tube 4 Wash the beads a b c d e Add 200 uL of SureSelect Binding Buffer Mix the beads on a vortex mixer for 5 seconds Put the tubes into a magnetic device such as the Dynal magnetic separator Life Technologies Remove and discard the supernatant Repeat step a through step d for a total of 3 washes 5 Resuspend the beads in 200 uL of SureSelect Binding Buffer SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing CAUTION Hybridization Step 3 Select hybrid capture with SureSelect Use these reagents from the SureSelect Target Enrichment Kit Box 1 SureSelect Wash 1 SureSelect Wash 2 SureSelect Elution Buffer SureSelect Neutralization Buffer Keep the Elution Buffer container tightly sealed when not in use Prolonged exposure of SureSelect Elution Buffer to air can decrease product performance by altering the pH of the solution 1 Estimate and record the volume of hybridization that remained after 24 hour incubation 2 Keep the PCR plat
48. s in Commercial Computer Software or Com puter Software Documentation Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met 2 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing In this Guide This guide describes the recommended operational procedures to capture transcripts of interest using the Agilent SureSelect RNA Capture Kit and sample preparation kits for next generation sequencing This protocol is specifically developed and optimized to use Biotinylated RNA oligomer libraries or Bait to enrich targeted regions of the transcriptome This guide uses the Illumina paired end multiplex sequencing platform for library preparation 1 Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand bef
49. s with overage at each step without the cDNA sample Master mixes for preparation of 12 samples including excess are shown in each table as an example Prepare the master mix on ice Use the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S 1 Preheat one heat block to 20 C and the other heat block to 37 C 2 For 1 library prepare on ice In a sterile microcentrifuge tube prepare the reaction mix in Table 18 Mix well by gently pipetting up and down 3 For multiple libraries prepare on ice a Prepare the reaction mix in Table 18 Mix well on a vortex mixer b Add 50 uL of the reaction mix to each 1 5 mL microcentrifuge tube c Add 50 uL of each DNA sample to each tube Mix by pipetting Change pipette tips between samples 4 Incubate in a thermal cycler for 30 minutes at 20 C Do not use a heated lid SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Table 18 End Repair Mix Sample Preparation Step 7 Repair the ends Reagent Volume for 1 Library Volume for 12 Libraries includes excess Purified double stranded cDNA sample 50 pL Nuclease Free Water 25 uL 342 5 uL Phosphorylation Reaction Buffer 10X 10 uL 125 pL Deoxynucleotide Solution Mix 10 mM 4 uL 20 uL each dNTP T4 DNA Polymerase 5 uL 62 5 uL DNA Polymerase Large Klenow Tul 12 5 uL Fragment T4 Polynucleotide Kinase 5 uL 62 5 uL p n E61008 Included in t
50. se free water to make enough mix for 2 uL per capture library plus excess d Add 2 uL of diluted SureSelect RNase Block purple cap to each capture library and mix by pipetting 6 Mix the contents in Table 25 to make the correct amount of SureSelect Block mix for the number of samples used SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 45 3 Hybridization Table 25 SureSelect Block Mix Reagent Volume for 1 reaction Volume for 12 reactions includes excess SureSelect Indexing Block 1 green cap 2 5 uL 31 25 uL SureSelect Block 2 blue cap 2 5 uL 31 25 uL SureSelect Indexing Block 3 brown cap 0 6 uL 7 5 yL Total 5 6 uL 70 uL 7 Inaseparate PCR plate prepare the prepped library for target enrichment a Add 3 4 uL of 30 ng uL prepped library to the B row in the PCR plate Put each sample into a separate well b Add 5 6 uL of the SureSelect Block Mix to each well in row B c Mix by pipetting up and down d Sealthe wells of row B with caps and put the PCR plate in the thermal cycler Do not heat the Hybridization Buffer or capture library yet only the prepped library with blockers e Start the thermal cycler program in Table 26 Figure 5 Prepped library shown in red Prepped Library 46 SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Hybridization 3 Table 26 PCR program Step Temperature Time Step 1 95 C 5 minut
51. supernatant e Repeat step a through step d for a total of 3 washes Make sure all of the wash buffer has been removed 10 Mix the beads in 50 uL of SureSelect Elution Buffer on a vortex mixer for 5 seconds to resuspend the beads 11 Incubate the samples for 10 minutes at room temperature 12 Separate the beads and buffer on a magnetic separator 13 Use a pipette to transfer the supernatant to a new 1 5 mL microfuge tube The supernatant contains the captured DNA The beads can now be discarded 14 Add 50 uL of SureSelect Neutralization Buffer to the captured DNA SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Hybridization 3 Step 4 Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color Do not freeze 3 Add 180 uL of homogenous AMPure XP beads to a 1 5 mL LoBind tube and add the 100 uL of cDNA library Mix well on a vortex mixer and incubate for 5 minutes 4 Put the tube in the magnetic stand Wait for the solution to clear approximately 3 to 5 minutes 5 Keep the tube in the magnetic stand Do not touch the beads while you carefully discard the cleared solution from the tubes 6 Continue to keep the tube in the magnetic stand while you dispense 0 5 mL of 70 ethanol in each tube Use fresh 70 ethanol for optimal result 7
52. the fragmented mRNA using reagents from the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100 As an alternative you can use the RNeasy MinElute Cleanup Kit Elute the RNA in 13 5 uL of Nuclease Free Water or elution buffer 1 Transfer the solution to a 1 5 mL RNase free non sticky tube 2 Add the components in Table 13 to the tube and incubate at 80 C for 30 minutes or overnight as desired Table 13 Component Volume 3 M NaDAC pH 5 2 2 uL Linear Acrylamide 10 mg mL 1 to 2 pL 100 ethanol 60 uL Included in the NEBNext mRNA Sample Prep Reagent Set 1 New England Bioscience p n E6100S Stopping Point You can safely stop the protocol here Store the samples at 15 C to 25 C Do not stop the protocol at any other point while the sample is RNA 3 Spin the tube in a microcentrifuge at 14 000 rpm 20 200 relative centrifugal force for 25 minutes at 4 C 4 Carefully pipette off the ethanol without dislodging the RNA pellet The RNA pellets are small and almost colorless To avoid dislodging the pellets remove the ethanol in several steps Remove 90 at each step and switch to smaller pipette tips for each step Without disturbing the pellet wash the pellet with 300 uL of 70 ethanol Spin the pellet in a centrifuge and carefully pipette out the 70 ethanol Air dry the pellet for 10 minutes at room temperature on c c Resuspend the RNA in 13 5 uL of Nuclease free water SureSele
53. ture Equipment Combinations Table 43 lists combinations of thermal cycler lid temperature plates or strips and sealing method strip caps or sealing tape other than those used in this protocol that have shown minimal evaporation Refer to this list for additional of equipment combination options for hybridization Note that minimal evaporation is needed to ensure good capture results Table 43 Tested options that show minimal evaporation PCR Machine Plate Strips Cover Comments Agilent Mx3005P Mx3000P Strip Tubes MX3000P Optical Strip Heated Lid QPCR 401428 Caps 401425 Agilent Mx3005P MicroAmp Optical MicroAmp Clear Heated Lid QPCR 96 well reaction plate Adhesive Film ABI compression pad N801 0560 4306311 4312639 Use two layers of film ABI GeneAmp 9700 MicroAmp Optical MicroAmp Caps Heated Lid 96 well Reaction Plate 8caps strip N801 0560 N801 0535 ABI Veriti 4375786 MicroAmp Optical MicroAmp Clear Heated Lid 96 well Reaction Plate Adhesive Film ABI compression pad N801 0560 4306311 4312639 Use two layers of film Eppendorf Eppendorf 8 Tube PCR Attached lids Lid heating set to 75 C Mastercycler Tubes BioRad MJ Agilent strip tubes Agilent Optical cap Heated Lid Research PTC 200 410022 Mx4000 410024 Mx4000 BioRad MJ Agilent strip tubes Agilent Optical cap Heated Lid Research PTC 200 410022 Mx4000 401425 Mx3000 3005 BioRad MJ Agilent 96 well Plate Agilent Optical cap Heated Lid Res
54. uded in the kit to adjust the pH to the proper range 4 Put a QIAquick spin column in a 2 mL collection tube 5 Transfer the 300 uL sample to the QIAquick spin column Spin the sample in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 6 Add 750 uL of Buffer PE concentrate to the column Spin the sample ina centrifuge for 60 seconds at 17 900 x g 13 000 rpm Discard the flow through 7 Put the QIAquick spin column back in the collection tube 2 mL and spin in a centrifuge for 60 seconds at 17 900 x g 13 000 rpm 8 Transfer the QIAquick spin column to a new 1 5 mL microcentrifuge tube to elute the cleaned sample 9 Let sit for 2 minutes to completely evaporate residual ethanol All traces of ethanol must be removed 10 Add 29 uL of Buffer EB directly onto the QIAquick filter membrane 11 Wait 60 seconds then centrifuge for 60 seconds at 17 900 x g 13 000 rpm 12 Collect the eluate Stopping Point If you do not continue to the next step store the samples at 20 C SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 33 2 Sample Preparation CAUTION Step 15 Amplify adaptor ligated library This step uses PCR to selectively enrich those cDNA fragments that have adaptor molecules on both ends and to amplify the amount of cDNA in the library The PCR is done with two primers that anneal to the ends of the adaptors Ten to fourteen cycles of PCR are used This
55. ureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing 63 4 Addition of Index Tags by Post Hybridization Amplification 64 4 Proceed to template denaturation and flow cell preparation Refer to the appropriate Illumina protocol Exact library pool dilution and processing can vary based on the flow cell capacity and analysis pipeline versions being used Refer to the appropriate Illumina user guide for instructions This protocol has been validated with 36 base paired end reads However read length can be adjusted to achieve the desired research goals SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification 4 Step 6 Prepare sample for cluster amplification In this step you set up cluster amplification Conditions are optimized to provide 700K to 900K clusters mm on the GAIIx and 400K to 600K clusters mm on a HiSeq instrument Genome Analyzer IIx Use reagents from the TruSeq Cluster Generation Kit appropriate for your instrument HT1 Hybridization Buffer HP3 2 N NaOH 1 Dilute 30 fmol 8uL of the 10 nM multiplexed sample pool with 16 uL of Buffer EB 10mM Tris Cl ph 8 5 for a total volume of 19 uL Add 1 uL of HP3 2 N NaOH Mix the sample briefly on a vortex mixer and pulse centrifuge Incubate for 5 minutes at room temperature to denature the DNA Place the sample on ice until you are ready to proceed to f
56. zer and start the run within five minutes after preparation 5 Within the instrument context choose the appropriate assay from the drop down list 6 Start the run Enter sample names and comments in the Data and Assay context 7 Verify the results Determine the concentration of the sample by integration under the peak You can use the High Sensitivity Kit to quantify the amount of sample to be used for Illumina sequencing The linear range of the High Sensitivity kit is 5 pg to 500 pg If the reading far exceeds 500 pg dilute and run the Bioanalyzer chip again If the yield is too low or non specific peaks are observed in the electropherogram repeat the PCR with more or fewer cycles The goal is to minimize cycles while you produce enough library for the quantification needed for application to the flow cell SureSelect RNA Target Enrichment for Illumina Paired End Multiplexed Sequencing Addition of Index Tags by Post Hybridization Amplification Step 3 Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 8 Continue to sequencing FU 300 250 200 150 100 Figure 8 5 10 150 200 300 400 S00 600 1000 200 1030 bp Analysis of Amplified Capture DNA using the High Sensitivity DNA Kit The electropherogram shows a single peak in the size range of approximately 250 to 300 20 nucleotides SureSelect RNA Target Enrichment for Illumina Paired End Multipl
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