SMARTer™ Ultra Low RNA Kit for Illumina Sequencing
Contents
1. 250 mM Tris HCl pH 8 3 375 mM KCI 30mM MgCl e 40ul dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM e 10 ul Dithiothreitol DTT 100 mM e 20ul SMARTScribe Reverse Transcriptase 100 U ul 1ml Nuclease Free Water e 655 ul RNase Inhibitor 40 U ul e 1ml Dilution Buffer 1 ml Purification Buffer 10 mM Tris Cl pH 8 5 Storage Conditions e Store Control Total RNA and SMARTer IIA Oligonucleotide at 70 C e Store Dilution Buffer at 4 C e Store all other reagents at 20 C www clontech com Protocol No PT5163 1 Version No PROX3693 3 SMARTer Ultra Low RNA Kit for Illumina Sequencing ll Additional Materials Required The following reagents are required but not supplied These materials have been validated to work with this protocol Please do not make any substitutions because you may not obtain the expected results Protocol No PT5163 1 Version No 4 PROX3693 Single channel pipette 10 ul 20 ul and 200 ul one each Eight channel pipette 20 ul and 200 ul one each Filter pipette tips 10 ul 20 ul and 200 ul one box each One QuickSpin minicentrifuge for 1 5 ml tubes One QuickSpin minicentrifuge for 0 2 ml tubes One marker pen Small 1 5 ml tube rack One bag of 8 strip nuclease free 0 2 ml thin wall PCR tubes with caps DNA OFF Solution Takara Cat No 9036 For PCR Amplification amp Validation One dedicated PCR thermal cycler used only for first strand synthesis A2. Prior to generating the final library for Illumina sequencing the Covaris AFA system is used for controlled DNA shearing The resulting DNA will be in the 200 500 bp size range 1 Turn power ON for the Covaris system and the main cooler Add about 1 9 L of distilled or deion ized water to the water bath The water level in the cooler should be within 3 mm of the FULL waterline when the transducer is submerged If needed add distilled or deionized water to the water bath until the FULL line is reached IMPORTANT Never run a process without the water bath This will permanently damage the transducer 2 Close the door and open the Sonolab software Click ON for the degassed button and degas the water bath for 1 2 hour 30 minutes 3 Add 65 ul of Purification Buffer to the DNA from Section V A Step 12 Transfer 75 ul of the Purifica tion Buffer DNA mixture into the 100 ul Covaris tube Put the sample tubes into the appropriate location on the Sample holder 4 Setup the process configuration panel based on the following table Table Ill Process Configuration Panel Set Up Duty Intensity Burst cycle Time min Mode 10 5 200 5 min Frequency Sweeping 5 Save the file and click return to go back to the main page 6 Open the door Place the tube holder with sample tubes on the transducer positioning system 7 Close the door 8 Click START on the main page to run the process 9 After s
3. User Manual SMARTer Ultra Low RNA Kit for Illumina Sequencing User Manual O Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Cat No 634935 Europe PT5163 1 PROX3693 33 0 1 3904 6880 Japan 81 0 77 543 6116 March 31 2011 Clontech Laboratories Inc ATakara Bio Company 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com SMARTer Ultra Low RNA Kit for Illumina Sequencing Table of Contents lL EiSts Of COnm POmernts ois sceccts cs cock cca sedck cans sac teeta cheba eat bade d eet deck eine eect eaten cece ve feet 3 Il Additional Materials Required ccccccceeeeeeeeeeneeeeeeeeeeeeeeeeeeeaeeaeeeseeeeeeeeeseeeseaaeeaeeeseeeeeeeeeens 4 MIN Mt OG UCHION svsisceveees csceeivinsss coves cence ctencesess tevevedenceveeraase iaaeao ran eara TKA aUa iadaa aeiia 5 IV SMARTer cDNA Synthesis o cccccccssccciveseaseciectcassceevenssccteedsdvaeees des sebevees a setveed Maieeiestsecbnedessvee 7 A Requirements for Preventing Contamination cccsessesssseseseseteeececsesceessesescsessescsessesecasacetenaeaenees 7 IB General Req mite ments rssssscsececscsecetiesesiesssed sacs cussxcsssecsscassseostipuvieyenses Seaver teagdiees e Eee RE RAER EAEE 7 G Sample Recommendations vcices iestirviecidh abs viesveses eect ncceteesete ts Mivetieiatb EA EEEE EEEE EEEE E EE 8 Di Sample Requirements niisisin sihasnaiisiin asin sna dewauervr
4. Any cell that has gone through fixation won t work the RNA won t release efficiently 1 Pick cell s in 1 ul of validated media see Section A Screen for Cell Culture Media then transfer to a 0 2 mL RNase free PCR tube containing 2 5 ul of Reaction Buffer Put the tube s on dry ice im mediately NOTE The total volume of suspended cells should not exceed 1 2 ul If necessary you can spin down your cells and resuspend them in Reaction Buffer e If you choose to transfer your cell s in 2 ul of media you need to use double the amount of Reaction Buffer 5 ul You must also double the volumes for all of the components in the first strand synthesis reaction Section IV E Steps 1 2 amp 4 as well as double the volume of beads for the first SPRI purification Section IV F Step 1 2 Proceed with Section IV E PROTOCOL First Strand cDNA Synthesis If you will not be performing cDNA synthesis immediately store the tube on dry ice or at 80 C till use Clontech Laboratories Inc www clontech com Protocol No PT5163 1 ATakara Bio Company Version No PROX3693 19 SMARTer Ultra Low RNA Kit for Illumina Sequencing IX Appendix C PCR Optimization Protocol No PT5163 1 Version No 20 PROX3693 If you have a sufficient amount of starting material gt 1 ng total RNA we recommend optimizing the PCR cycling parameters for your experiment If you have a very limited amount of material or your sample is unique
5. Sample 1 ul Total Volume 3 5 ul 3 5 ul 3 5 ul The Control RNA is supplied at a concentration of 1 ug ul The Control RNA should be diluted in nuclease free water to match the concentration of your test sample Perform serial dilutions on the Control RNA until you obtain the appropriate concentration 3 Place the samples on a 20 C prechilled IsoFreeze PCR rack in a PCR clean station and add 1 ul of 3 SMART CDS Primer II A 12 uM Mix the contents and spin the tube s briefly in a microcentrifuge 3 5 ul Cell Total RNA in Reaction Buffer from Table l 1 ul 3 SMART CDS Primer II A 12 uM Recipe 4 5 ul Total Volume 4 Incubate the tube s at 72 C in a hot lid thermal cycler for 3 min then put the samples on the IsoFreeze PCR rack NOTE The initial reaction steps Step 5 7 are critical for first strand synthesis and should not be delayed after Step 4 You can prepare your master mix for Step 5 while your tubes are incubating Step 4 in order to jump start the cDNA synthesis 5 Meanwhile prepare a Master Mix for all reactions plus one by combining the following reagents in the order shown at room temperature 2 ul 5X First Strand Buffer 0 25 ul DTT 100 mM 1 ul dNTP Mix 10 mM 1 ul SMARTer IIA Oligonucleotide 12 uM Recipe 0 25 ul RNase Inhibitor 1 ul SMARTScribe Reverse Transcriptase 100 U 5 5 ul Total Volume added per reaction Add the reverse transcriptase
6. RNA used in the first strand synthesis IMPORTANT Optimal parameters may vary with different templates and thermal cyclers To determine the optimal number of cycles for your sample and conditions we strongly recommend that you perform a range of cycles Do not exceed the recommended cycle numbers in the table below for different starting amounts of material Refer to Appendix C for PCR optimization guidelines Table II Cycling Guidelines Based on Amount of Starting Material Input Amount Input Amount Typical No of Total RNA Cells PCR Cycles 10 ng 1000 cells 12 1ng 100 cells 12 500 pg 50 cells 13 100 pg 10 cells 15 www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing IV SMARTer cDNA Synthesis continued 1 Prepare a PCR Master Mix for all reactions plus one additional reaction Combine the following reagents in the order shown then mix well by vortexing and spin the tube briefly in a microcentrifuge 5 ul 10X Advantage 2 PCR Buffer 2 ul dNTP Mix 10 mM 2 ul IS PCR Primer 12 uM Recip 2 ul 50X Advantage 2 Polymerase Mix 39 ul Nuclease Free Water 50 ul Total Volume per reaction 2 Add 50 ul of PCR Master Mix to each tube containing DNA bound to the beads from Section IV F Step 4 Mix well and briefly spin down IMPORTANT hd Transfer the samples from the PCR Clean Work Station to the general lab All downstre
7. Troubleshooting Guide for SMARTer cDNA Synthesis amp Amplification sseseseeeseereerereerersee 21 Contact Us For Assistance Customer Service Ordering Technical Support Telephone 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 650 424 1064 Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Protocol No PT5163 1 www clontech com Clontech Laboratories Inc Version No PROX3693 ATakara Bio Company 2 SMARTer Ultra Low RNA Kit for Illumina Sequencing I List of Components Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing The following components have been specifically designed to work together and are optimized for this particular protocol Please do not make any substitutions The substitution of reagents in the kit and or a modification of the protocol may lead to unexpected results Box 1 e 204ul SMARTer II A Oligonucleotide 12 uM 5 AAGCAGTGGTATCAACGCAGAGTACXXXXX 3 X undisclosed base in the proprietary SMARTer oligo sequence e 54 Control Total RNA 1 yg ul Box 2 e 20ul 3 SMART CDS Primer Il A 12 uM 5 AAGCAGTGGTATCAACGCAGAGTACT N A C G orT N A G or C N_ N 3 30 e 204ul IS PCR Primer 12 uM 5 AAGCAGTGGTATCAACGCAGAGT 3 Please do not substitute The oligo has been modified e 40ul 5X First Strand Buffer RNase Free
8. are added in a right order Failure of any components of the reaction will result in a failed cDNA synthesis Make sure to prepare all the necessary items prior cDNA synthesis and to spin down tubes prior opening them Do not delay cDNA synthesis reaction after RNA is denatured to jump start cDNA synthesis PCR failed cDNA was not efficiently bound to the SPRI beads during purification steps Make sure to use the recommended PCR enzyme Before using the PCR enzyme check it with previ ously tested primers and template Make sure beads are mixed correctly after adding them to DNA samples Vortexing the beads once they are added to samples can shear the DNA or break it free from the beads Make sure the SPRI beads are collected correctly on the magnetic stand and given enough time to completely separate the liquid phase Make sure not to disturb the beads while removing the supernatant Supernatant that is not completely removed from SPRI beads may inhibit downstream reactions RNA is low quality degraded or has impurities that inhibit the cDNA synthesis reaction If possible check RNA quality prior to cDNA syn thesis Make sure RNA is good quality Certain tis sues such as plants have a high level of polysac charides that interfere with first strand synthesis Make sure to use appropriate RNA isolation kits for each given tissue species Control RNA is too diluted Make sure to use recently calibr
9. beads NOTE The beads are viscous suck the entire volume up and push it out slowly Place the 96 well plate on the Ambion Magnetic Stand 96 for 5 min or longer until the liquid ap pears completely clear and there are no beads left in the supernatant While the plate is sitting on the magnetic stand pipette out the supernatant Keep the plate on the magnetic stand Add 200 ul of freshly made 80 Ethanol to each sample without disturbing the beads to wash away contaminants Wait for 30 seconds and carefully pipette out the supernatant DNA will remain bound to the beads during the washing process Repeat Step 5 one more time Seal the sample wells on the plate and briefly spin down for 10 seconds at 1 000 rpm to collect the liquid at the bottom of the well Place the 96 well plate on the magnetic stand for 30 seconds then remove all the remaining Ethanol Place the plate at room temperature for 3 5 min until the pellet appears dry You may see a tiny crack in the pellet NOTE If you over dried the beads you will see many cracks in the pellet If it is under dried the DNA recovery rate will be lower because of the remaining Ethanol 10 Once the beads are dried add 12 ul of Purification Buffer to cover the beads Remove the plate from the magnetic stand and incubate at room temperature for 2 min to rehydrate 11 Mix the pellet by pipetting up and down 10 times to elute DNA from the beads then put the plate back on the
10. to the master mix just prior to use Mix well by gently vortexing and spin the tube s briefly in a microcentrifuge 6 Add 5 5 ul of the Master Mix to each reaction tube from Step 4 Mix the contents of the tube s by gently pipetting and spin the tube s briefly to collect the contents at the bottom 7 Incubate the tubes at 42 C for 90 min 8 Terminate the reaction by heating the tube s at 70 C for 10 min Protocol No PT5163 1 Version No PROX3693 9 Clontech Laboratories Inc www clontech com ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing IV SMARTer cDNA Synthesis continued P PROTOCOL P PROTOCOL e Important Protocol No PT5163 1 Version No PROX3693 10 F PROTOCOL Purification of First Strand cDNA using SPRI Ampure Beads perform in PCR Clean Work Station The first strand cDNA selectively binds to SPRI beads leaving contaminants in solution which is re moved by a magnetic separation The beads are then directly used for PCR amplification NOTE e Before use beads should be brought to room temperature and mixed well to disperse e We recommend taping a 10 ul pipette tip refill rack onto the top of the MagnaBot Magnetic Separation device to keep the tubes strips in place To purify the SMART cDNA from unincorporated nucleotides and small lt 0 1 kb cDNA fragments follow this procedure for each reaction tube 1 Add 25 ul of SPRI Ampure XP bea
11. use a similar source of RNA or cells to perform PCR cycle optimization prior to using the actual sample Choosing the optimal number of PCR cycles ensures that the ds cDNA will remain in the exponen tial phase of amplification When the yield of PCR products stops increasing with more cycles the reaction has reached its plateau Overcycled cDNA can result in a less representative sample Undercycling on the other hand results in a lower yield of cDNA The optimal number of cycles for your experiment is one cycle fewer than is needed to reach the plateau Be conservative when in doubt it is better to use fewer cycles than too many To perform PCR cycle optimization prepare several tubes containing an amount of RNA equal to your sample amount Subject each tube to a different range of cycles For example if you have 1 ng of RNA subject one tube to the recommended a number of cycles Subject the other two tubes to 2 3 cycles fewer or more than the first tube i e 15 12 recommended for 1 ng and 10 cycles 1 Use the following program for thermal cycling e 95 C 1 min e X cycles 95 C 15sec 65 C 30sec 68 C 6 min e 72 C 10min e 4C forever 2 Perform Purification of ds cDNA using SPRI Ampure Beads Section V A 3 Run samples on an Agilent High Sensitivity DNA Chip using the Agilent 2100 Bioanalyzer to evaluate DNA output See the user manual for the Agilent High Sensitivity DNA Kit for instructions http www chem agilent com
12. you are using Low cDNA yield with the experimental RNA but control is OK XI References Protocol No PT5163 1 Version No 22 PROX3693 Barnes W M 1994 PCR amplification of up to 35 kb DNA with high fidelity and high yield from bacteriophage tem plates Proc Natl Acad Sci USA 91 2216 2220 Chenchik A Zhu Y Diatchenko L Li R Hill J amp Siebert P 1998 Generation and use of high quality cDNA from small amounts of total RNA by SMART PCR In RT PCR Methods for Gene Cloning and Analysis Eds Siebert P amp Larrick J BioTechniques Books MA pp 305 319 Kellogg D E Rybalkin I Chen S Mukhamedova N Vlasik T Siebert P amp Chenchik A 1994 TaqStart Antibody Hot start PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase BioTechniques 16 1134 1137 Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc Your use of this productis also subject to compliance with any applicable licensing requirements described on the product s web page at http w
13. 1 ATakara Bio Company Version No PROX3693 17 SMARTer Ultra Low RNA Kit for Illumina Sequencing VIII Appendix B Working with Cells e mportant Protocol No PT5163 1 Version No PROX3693 18 A PROTOCOL Screen for Cell Culture Media IMPORTANT When working with cultured cells you need to ensure that the cell culture medium does not inhibit first strand cDNA synthesis Therefore it is important to select a medium with the least inhibitory effect Please select cell culture medium according to the protocol below Use the selected medium to perform serial dilution of your cells and picking of cells for cDNA synthesis 1 Prepare 10 ng ul amp 100 pg ul serial dilutions of the Control Total RNA and cell sample in Nuclease Free Water then dilute to 5 pg ul with Reaction Buffer for the positive control or test medium for the test sample Collect as many media as possible in order to do one screening with a negative and positive control per test sample See Table IV for guidelines Table IV Media Screen Preparation Guidelines Components Negative Control Positive Control Test Sample Reaction Buffer 2 5 ul Nuclease Free Water 1 yl 1 ul Total RNA Cell Suspension 2 5 ul 5 pg ul 2 5 ul 5 pg ul Test Buffer Medium 1 ul Total Volume 3 5 ul 3 5 ul 3 5 ul 2 Perform the SMARTer cDNA synthesis protocol Section IV E G with samples and controls from Step 1 using 18 cycles
14. 5 100 200 300 400 500 700 2000 10380 bp B Ful nc 18 1004 amp TET ERTL T TEUER A SEEL l EPA EAETUTAU 35 100 200 300 400 600 1000 3000 10380 bp Figure 3 Electropherogram example results from Agilent 2100 Bioanalyzer All samples were subjected to SMARTer cDNA synthesis and amplification as described in the protocol FU fluorescence absorption units Panel A Clean SMARTer Amplification Product 15 PCR cycles Panel B Clean SMART Negative Control 18 PCR cycles Panel C see pg 14 Clontech Laboratories Inc www clontech com Protocol No PT5163 1 ATakara Bio Company Version No PROX3693 13 SMARTer Ultra Low RNA Kit for Illumina Sequencing V Amplified cDNA Purification amp Validation continued c FU 7 sample 3 1604 1404 1204 1004 80 4 60 7 40 20 20 35 100 200 300 400 600 1000 3000 10380 bp Figure 3 continued Electropherogram example results from Agilent 2100 Bioanalyzer Sample was subjected to SMARTer cDNA synthesis and amplification as described in the protocol Panel C Example of Contaminated SMARTer Amplification Product Protocol No PT5163 1 Version No PR0X3693 14 www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing VI Covaris Shearing P PROTOCOL e Important Clontech Laboratories Inc ATakara Bio Company A PROTOCOL Covaris Shearing of Full length cDNA
15. ATakara Bio Company A Requirements for Preventing Contamination Before you set up the experiment make sure you have two physically separated work stations The first should be the PCR Clean Work Station see Appendix A for all Pre PCR experiments that require clean room conditions such as first strand cDNA synthesis Section IV E and purification of first strand cDNA Section IV F The second work station can be in the general laboratory where you will perform PCR measure cDNA concentration and work on any other experiments involving the PCR amplified cDNA It is absolutely required to have a PCR work station in a clean room with positive air flow as con tamination may occur very easily Once contamination occurs it can be difficult to remove You need to strictly follow the clean room operation rules i e you can only move materials sup plies from the clean room to the general lab NOT the other way around Don t share any equipment reagents between the clean room and the general lab You need a separate PCR machine inside the PCR workstation for cDNA synthesis Wear gloves and sleeve covers throughout the procedure to protect your RNA samples from degra dation by contaminants and nucleases Be sure to change gloves and sleeve covers between each section of the protocol B General Requirements The success of your experiment depends on the quality of your starting sample of RNA Prior to cDNA synthesis please make sure that
16. Library usermanuals Public G2938 90320_HighSensitivityDNAQuick pdf 4 Determine the optimal number of cycles required for each experimental and control sample We recommend using the lowest PCR cycle number that generates enough material for Illumina library construction 5 Apply the optimal number of PCR cycles to your sample material www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing X Appendix D Troubleshooting Guide Clontech Laboratories Inc ATakara Bio Company Table VI Troubleshooting Guide for SMARTer cDNA Synthesis amp Amplification PROBLEM No cDNA yield with the control RNA No cDNA yield with the experimental RNA but control is OK Low cDNA yield with the control RNA Control RNA is degraded SOLUTION The control RNA is vigorously tested for RNase con taminations and provided in high concentrations 1 ug ul to reserve its integrity It is essential that any item that could contact the control RNA is RNase free Control RNA is too diluted Make sure to use recently calibrated pipettes to ac curately perform serial dilutions of the control RNA Prepare fresh dilutions of the Control RNA and try again The diluted RNA is less stable therefore avoid using previously diluted low concentration RNA samples cDNA synthesis reaction failed Carefully check the protocol and make sure all components
17. Sequencing lll Introduction continued Protocol No PT5163 1 Version No 6 PROX3693 SMARTer cDNA synthesis starts with picogram amounts of total RNA A modified oligo dT primer the SMART CDS Primer primes the first strand synthesis reaction Figure 2 When SMARTScribe Reverse Transcriptase reaches the 5 end of the mRNA the enzyme s terminal transferase activity adds a few ad ditional nucleotides to the 3 end of the cDNA The carefully designed SMARTer Oligonucleotide base pairs with the non template nucleotide stretch creating an extended template to enable SMARTScribe RT continue replicating to the end of the oligonucleotide Chenchik et al 1998 The resulting full length single stranded ss cDNA contains the complete 5 end of the mRNA as well as sequences that are complementary to the SMARTer Oligonucleotide In cases where the RT pauses before the end of the template the addition of non template nucleotides is much less efficient than with full length cDNA RNA hybrids thus the overhang needed for base pairing with the SMARTer Oligonucleotide is absent The SMARTer anchor sequence and the poly A sequence serve as universal priming sites for end to end cDNA amplification In contrast CDNA without these sequences such as prematurely terminated cDNAs contaminating genomic DNA or cDNA transcribed from poly A RNA will not be exponentially amplified However truncated RNAs with poly A tails that are present in poo
18. a ccccceccceceecseceecceccecsecevesceecsseveeesseessesenes 18 B Cell Preparation for First Strand CDNA Synthesis csceesssessscescsesseeeeceeseeeeeeseseacneeasecaeaeeesesatacsees 19 IX Appendix C PCR Optimization cccccccessssceeeesseneeeeeeeeeaeeeeeeseaaeeeeesseaeeeeseeneaeeeeeseeaaeeeeneee 20 X Appendix D Troubleshooting Guide ccccccccceeesssseceeeeeeseeeeeeeeseeeeeeesscaeeeeneesseseeeeeesaeaeenes 21 MU IREPERENIGCES oi scees a oes secestcesvaceatabedeenstaboccveeistiveviedsfesztecdzet tatvarss S 22 List of Figures Figure 1 Protocol Overview moeren E r N hiatal a anaemia entities 5 Figure 2 Flowchart of SMARTer cDNA Synthesis sssssesessesesseseeseeeteestesrsersrstrsrstsrrstssrsesntrsntrsnterenterenees 6 Figure 3 Electropherogram example results from Agilent 2100 Bioanalyzer ssssssesssresssesesseserrerereerereee 13 List of Tables Table I Sample Preparation Guidelines s ssssssessssessssesssssesterestetstertetttetisierrsrerrstsrrstnnrstnntntenterentnrenterentet 9 Table II Cycling Guidelines Based on Amount of Starting Material eseseesenseeeeceeseeteeeeseeeneeaes 10 Table HI Process Configuration Panel Set Up eessesscsssssesecseseeeseeseensecesescaseeseceeaesesesaeaeeesassenseneseeaes 15 Table IV Media Screen Preparation Guidelines cceccssssseeseseeeseesesesecesesenseesecseaeeesesaeseeeeanecaseeeeseeaes 18 Table V Validated Media rcns aE ain EAA EEEIEE EEE 18 Table VI
19. aia E ni NEEE EEEE Sis 8 E PROTOCOL First Strand CDNA Synthesis sscscsssssssscscsssssssscesssscsscesssssesssssssessssssssesessseseessssessess 8 F PROTOCOL Purification of First Strand cDNA using SPRI Ampure Beads 0 0 eeseessseeeeeereeeeees 10 G PROTOCOL ds cDNA Amplification by LD PCR cssscssecscsscssssscsssesssssssessscssssssssessesseseeses 10 V Amplified cDNA Purification amp Validation cccccescceceessseeeeeeesseceeeesssneeeeseesneseeeeessneaeenes 12 A PROTOCOL Purification of ds CDNA using SPRI Ampure Beads 0 seeceesesseseecneeeeseeeseeeteeeeaees 12 B Validation Using the Agilent 2100 BioAnalyzer oo ccecesesssesseseecscseeeecseseeeseeseseseeseaeecssaeeesesataceees 12 VI Covaris SNe arin G sii ss iesseci cece teceiiiehssdernclds cashins canpudsshend EErEE EENT EEEN EEKE EEKEREN Ei 15 A PROTOCOL Covaris Shearing of Full length CDNA cee seseseeecessesesecesesenseesecseseeesesasseeeseeanes 15 VII Appendix A PCR Clean Work Station Guidelines c cccssssccceessseceeeeeesseeeeeeeesseeeenes 16 A Equipment Needed in the PCR Clean Work Station ciccseessessseeessesesensecssesenseesecseneeesesasseesenanes 16 B PCR Clean Work Station Operation Instructions c cccesesesesesseecsesseeeeceeseseseeseseaenseaeecaeneeesecateceees 16 VIII Appendix B Working with Cells cccccceeceeeseceeceeee eee eeeeeee eee aaeaaeeeseseeeeeeeseeseeeseaaeeeeeeees 18 A PROTOCOL Screen for Cell Culture Medi
20. am mportant processes will be performed in the general lab 3 Place the tube in a preheated thermal cycler with a heated lid Commence thermal cycling using the following program e 95 C 1 min e Xcycles 95 C 15 sec 65 C 30 sec 68 C 6 min e 72 C 10 min e 4 C forever Consult Table II for guidelines Determine the optimal number of PCR cycles using the protocol in Appendix C PCR Optimization Clontech Laboratories Inc www clontech com Protocol No PT5163 1 ATakara Bio Company Version No PROX3693 11 SMARTer Ultra Low RNA Kit for Illumina Sequencing V Amplified cDNA Purification amp Validation P PROTOCOL Protocol No PT5163 1 Version No 12 PR0X3693 A PROTOCOL Purification of ds cDNA using SPRI Ampure Beads PCR amplified cDNA is purified by immobilizing it onto SPRI beads The beads are then washed with 80 Ethanol and eluted in Purification Buffer 1 Take out a 96 well Axygen V bottom plate and cover all the wells with a MicroAmp Clean Adhesive Seal Uncover only the wells that you want to use Vortex SPRI beads till even then add 90 ul of SPRI Ampure XP Beads to the wells of the 96 well plate Transfer the entire PCR product including the SPRI beads from Section IV G Step 3 to the wells of the plate containing the SPRI beads from Step 1 above Pipette the entire volume up and down 10 times to mix thoroughly Incubate at room temperature for 8 min to let the DNA bind to the
21. aring your cells for first strand cDNA synthesis E PROTOCOL First Strand cDNA Synthesis perform in PCR Clean Work Station IMPORTANT To avoid introducing contaminants to your RNA sample the first part of the cDNA synthesis protocol Sections E G requires use of a PCR work station in a clean room Standard clean room procedure should be followed If no clean room is available you may work with just a PCR Clean Work Station on a temporary basis We strongly recommend putting the PCR work station in a clean room to avoid contamination It is critical to have an air blower in the PCR work station turned on during the whole process Refer to Appendix A for PCR Clean Work Station Guidelines 1 Prepare a stock solution of Reaction Buffer by mixing the Dilution Buffer with the RNase Inhibitor as indicated below scale up as needed 19 ul Dilution Buffer 1 ul RNase Inhibitor 20 ul Total Volume www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing IV SMARTer cDNA Synthesis continued 3 5 ul to individual 0 2 mL RNase free PCR tubes in an 8 well strip Table I Sample Preparation Guidelines 2 See Table for guidelines on setting up your control amp test samples Transfer each whole volume of Components Negative Control Positive Control Test Sample Reaction Buffer 2 5 ul 2 5 ul 2 5 ul Nuclease free water Tul Diluted Control RNA 1 ul
22. ated pipettes to ac curately perform serial dilutions of the control RNA Prepare fresh dilutions of the Control RNA and try again Diluted RNAisless stable therefore avoid using previously diluted low concentration RNA samples cDNA was not efficiently bound to the SPRI beads during purification steps Make sure the SPRI beads are collected correctly on the magnetic stand and given enough time to completely separate the liquid phase Make sure not to disturb beads while removing the super natant Repeat cDNA synthesis with the freshly diluted control RNA SPRI beads were overdried during purification of amplified cDNA www clontech com Don t allow the post wash bead pellet to be over dried It s important to allow any residual ethanol to dry but overdrying too long or drying at a tem perature higher than room temperature will make it difficult to elute the DNA from the beads Protocol No PT5163 1 Version No PROX3693 21 SMARTer Ultra Low RNA Kit for Illumina Sequencing X Appendix D Troubleshooting Guide continued Table VI Troubleshooting Guide for SMARTer cDNA Synthesis amp Amplification continued PROBLEM SOLUTION Insufficient amount of starting mate If more RNA sample is available quantitate rial or RNA is degraded or has impu RNA prior to cDNA synthesis Follow PCR cycle rities that inhibit the cDNA synthesis guidelines appropriate for the starting amount of reaction sample
23. ds to each sample using a 200 ul pipetter Adjust the pipetter to 35 ul and pipette the entire volume up and down 10 times to mix thoroughly The beads are viscous suck the entire volume up and push it out slowly Incubate at room temperature for 8 minutes to let DNA bind to the beads 2 Briefly spinthe sample tubes to collect the liquid from the side of the wall Place the sample tubes on the Promega MagnaBotll Magnetic Separation Device for5 min or longer until the solution is completely clear 3 While samples are still on the Magnetic Separation Device pipette out the solution and discard Briefly spin the tubes to collect the liquid at the bottom 4 Place the tubes back in the Promega MagnaBot II Magnetic Separation Device for 2 min or longer to let beads separate from the liquid completely Pipette out the residual liquid from the beads using a 10 ul pipetter and discard Make sure that there is no supernatant remaining in the tube Be careful not to take out any beads with the supernatant G PROTOCOL ds cDNA Amplification by LD PCR perform steps 1 amp 2 in PCR Clean Work Station We strongly recommend use of the Advantage 2 PCR Kit Cat Nos 639206 amp 639207 for PCR amplification The Advantage 2 Polymerase Mix has been specially formulated for efficient and accurate amplification of cDNA templates by long distance PCR LD PCR Barnes 1994 Table II provides guidelines for optimizing your PCR depending on the amount of total
24. dvantage 2 PCR Kit Cat Nos 639206 amp 639207 This kit has been specially formulated to provide automatic hot start PCR Kellogg et al 1994 and can efficiently amplify full length cDNAs with a significantly lower error rate than that of conventional PCR Barnes 1994 High Sensitivity DNA Kit Agilent Cat No 5067 4626 lsoFreeze Flipper Rack MIDSCI Cat No TFL 20 IsoFreeze PCR Rack MIDSCI Cat No 5640 T4 Nuclease free thin wall PCR tubes 0 2 ml USA Scientific Cat No 1402 4700 Nuclease free nonsticky 1 5 ml tubes USA Scientific Cat No 1415 2600 For SPRI Bead Purification Agencourt AMPure PCR Purification Kit 5 mL Beckman Coulter Part No A63880 60 mL Beckman Coulter Part No A63881 Use this kit for the SPRI Purifications Sections IV F amp V A MagnaBot II Magnetic Separation Device Promega Part No V8351 Use this stand for the first purification Section IV F Magnetic Stand 96 Ambion Part No AM10027 Use this stand for the second purification Section V A 96 well V bottom Plate 500 uL VWR Cat No 47743 996 MicroAmp Clean Adhesive Seal AB Part No 4306311 80 Ethanol For Sequencing Library Generation Illumina Paired End DNA Sample Prep Kit Illumina Cat Nos PE 102 1001 amp PE 102 1002 Covaris Instrument and Related Materials for DNA Shearing www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing l
25. e most traditional RNA isolation methods may not be applicable There are several commercially available products that enable purification of total RNA preparations from extremely small samples e g Clontech offers the NucleoSpin RNA XS Kit Cat No 740902 10 for purification of RNA from 10 cells When choosing a purification method kit ensure that it is appropriate for your sample amount Though SMART Technology is sensitive enough to generate cDNA from as little as 10 pg of total RNA the use of a higher amount of starting material 100 pg to 10 ng is recommended for reproducible am plification of low abundance mRNA transcripts After RNA extraction if your sample size is not limiting we recommend evaluating total RNA quality using the Agilent RNA 6000 Pico Kit Cat No 5067 1513 Sample Requirements Total RNA This protocol has been optimized for cDNA synthesis starting from 100 pg of total RNA However if your RNA sample is not limiting we recommend that you start with more total RNA up to 10 ng Purified total RNA should be in nuclease free water If the total RNA volume is larger than 1 ul perform ethanol precipitation and resuspend the pellet in 1 ul of nuclease free water Cells Although this protocol was optimized for cDNA synthesis starting from total RNA the protocol has also been validated to work starting from cells See Appendix B Working with Cells for instructions on select ing the appropriate media and prep
26. ents back in the 20 C freezer Close the cover window of the PCR clean work station leave the blower on and dispose of gloves and sleeve covers www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing VII Appendix A PCR Clean Work Station Guidelines continued Clean Work Station Instructions for Purification of fs cDNA amp LD PCR Sections IV F amp IV G 7 Bring the Ampure XP beads from 4 C to the PCR clean work station Aliquot the beads into 2 tubes one for the first strand cDNA purification Section IV F and one for the PCR product purification Section V A prior to the experiment to avoid a cross contamination 8 Bring the lsoFreeze Flipper Rack containing the SMARTer PCR reagents from the 20 C freezer to the PCR clean work station and put it on top of the kimwipes 9 Set up the PCR reactions 10 Take the PCR reaction tubes out of the PCR clean work station and perform PCR in a thermal cycler in the general lab 11 Place used tips and tubes in a trash bag remove from the PCR clean work station and dispose 12 Put the IlsoFreeze Flipper Rack containing the SMARTer PCR reagents back in the 20 C freezer and put the Ampure XP beads back in the 4 C refrigerator 13 Close the cover window of the PCR clean work station Turn off the light and blower and turn on the UV light Clontech Laboratories Inc www clontech com Protocol No PT5163
27. hearing is complete transfer 75 ul of sheared DNA to 1 5 ml tubes 10 Proceed to generate an Illumina Sequencing Library with the Illumina Paired End DNA Sample Prep Kit Illumina Cat Nos PE 102 1001 amp PE 102 1002 Dispose all tubes and pipettes that have been exposed to amplicons in a sealed trash bag www clontech com Protocol No PT5163 1 Version No PROX3693 15 SMARTer Ultra Low RNA Kit for Illumina Sequencing VII Appendix A PCR Clean Work Station Guidelines A Equipment Needed in the PCR Clean Work Station e Important Important Protocol No PT5163 1 Version No 16 PROX3693 IMPORTANT The following equipment must be kept in the PCR Clean Work Station only Don t share any equipment reagents between the clean room and the general lab You can only move materials supplies from the clean room to the general lab NOT the other way around Do not use equip ment from the general lab forthe portions of the protocol that require a PCR CleanWork Station Single channel pipette 10 ul 20 ul and 200 ul one each Eight channel pipette 20 ul and 200 ul one each Filter pipette tips 10 ul 20 ul and 200 ul one box each One dedicated PCR thermal cycler used only for first strand synthesis One QuickSpin minicentrifuge for 1 5 ml tube amp one QuickSpin minicentrifuge for 0 2 ml tube MagnaBot II Magnetic Separation Device Promega Part No V8351 e One marker pen Small 1 5 ml
28. ll Introduction SMARTer cDNA Synthesis for the Illumina Sequencing Platform The SMARTer Ultra Low RNA Kit allows high quality cDNA synthesis starting from as little as 100 pg of total RNA or cells The kit has been designed and validated to prepare cDNA samples for sequencing and quantitation with the Illumina HiSeq and Genome Analyzer sequencing instruments The entire library construction protocol can be completed within two days see Figure 1 for an overview of the proto col SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts enabling a direct start from your sample Most importantly SMART technology enriches for full length transcripts and maintains the true representation of the original mRNA transcripts these factors are criti cal for transcriptome sequencing and gene expression analysis Start with Total RNA or cells Section IV C SMARTer First strand cDNA Synthesis amp Purification Sections IV E amp IV F Full length ds cDNA Amplification by LD PCR Section IV G DAY ONE Amplified cDNA Purification amp Validation Section V A amp V B Covaris Shearing of Full length cDNA Section VI illumina Paired End DNA Sample Prep for Sequencing DAY TWO Cluster Generation Figure 1 Protocol Overview Clontech Laboratories Inc www clontech com Protocol No PT5163 1 ATakara Bio Company Version No PROX3693 5 SMARTer Ultra Low RNA Kit for Illumina
29. magnetic stand for 1 minute or longer until the solution is completely clear 12 Transfer clear supernatant containing purified cDNA from each well to a nuclease free nonsticky tube Label each tube with sample information and store at 20 C B Validation Using the Agilent 2100 BioAnalyzer 1 Aliquot 1 ul ofthe amplified cDNA for validation using the Agilent 2100 BioAnalyzer and the High Sensi tivity DNA Chip from Agilent s High Sensitivity DNA Kit Cat No 5067 4626 See the user manual forthe Agilent High Sensitivity DNA Kit for instructions http www chem agilent com Library usermanu als Public G2938 90320_HighSensitivityDNAQuick pdf Compare the results for your samples amp controls see Figure 3 to verify whether the sample is suit able for further processing Successful cDNA synthesis and amplification should yield no product in the negative control Figure 3 Panel B and a distinct peak spanning 400 bp to 9000 bp peaked at 2000 bp for the control RNA sample Figure 3 Panel A yielding approximately 2 7 ng of cDNA depending on the input Contaminated samples will have a broader peak and abnormally high yield Figure 3 Panel C Proceed to Section VI Covaris Shearing www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing V Amplified cDNA Purification amp Validation continued A Ful 100pg 15cyc 1404 1204 404 20 3
30. of PCR 3 Purify the PCR products with SPRI beads Section V A 4 Run 1 pl ofeach sample from Step 3 on an Agilent High Sensitivity DNA Chip using the Agilent 2100 Bioanalyzer See the user manual for the Agilent High Sensitivity DNA Kit for instructions http www chem agilent com Library usermanuals Public G2938 90320_HighSensitivityDNAQuick pdf 5 Compare the yield between samples diluted in culture media and nuclease free water Select the media that exhibits the least inhibitory effects for cell picking Table V Validated Media Superblock Pierce Cat No 37515 0 1 mL of DMEM F12 Glutamax Invitrogen Cat No 10565 3 6 ul of 25 BSA Invitrogen Cat No A10008 01 For 1 L of PBS Buffer 0 2 micron filtered 0 2 g of KCI 0 24 g of KH PO anhydrous 8 00 g of NaCl 1 44 g of Na HPO anhydrous add dH O to 1 L We recommend testing the viability of your cells in the media listed in Table V these media have been tested with this protocol If your cells do not thrive in any of the media listed in Table V screen other media per the instructions listed above www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing Vill Appendix B Working with Cells continued B Cell Preparation for First Strand cDNA Synthesis IMPORTANT The cDNA synthesis protocol has been tested with suspension cells without internal label mportant ing
31. r quality RNA starting material will be amplified yielding shorter cDNA fragments Poly At RNA DS ADDDDDNPPPVy polyA 3 KK 5 AA u x SMARTer II A SMART CDS primer Oligonucleotide First strand synthesis amp tailing Single by SMARTScribe RT step AA 5 AAAA polyA 5 rhs Template switching and extension by SMARTScribe RT x yok WSSSSSSSIY polyA y a S S a Amplify cDNA by LD PCR with IS PCR primer Double stranded cDNA ME MM cr ee Figure 2 Flowchart of SMARTer cDNA Synthesis The SMARTer II A Oligonucleotide 3 SMART CDS Primer II A and IS PCR Primer all contain a stretch of identical sequence see Section for sequence information www clontech com Clontech Laboratories Inc ATakara Bio Company SMARTer Ultra Low RNA Kit for Illumina Sequencing IV SMARTer cDNA Synthesis PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING This protocol is optimized for the generation of cDNA starting from ultra low amounts of total RNA using Clontech s SMART technology The protocol also works starting from cells Due to the sensitivity of the protocol the input material total RNA or cells needs to be collected and purified under clean room conditions to avoid contamination The whole process of SMARTer cDNA Synthesis should be carried out in a PCR Clean Work Station under clean room condi tions see Appendix A PCR Clean Work Station Guidelines Important Clontech Laboratories Inc
32. tube rack Small bottle of nuclease free water e One bag of 1 5 ml nuclease free tubes One bag of 8 strip nuclease free 0 2 ml thin wall PCR tubes with caps DNA OFF Solution Takara Cat No 9036 B PCR Clean Work Station Operation Instructions IMPORTANT At the beginning of each section of the protocol listed below be sure to put on aclean pair of gloves and sleeve covers then turn on the light and blower on the PCR clean station and lift the cover window Bring a clean trash bag into the PCR clean station as well as two pieces of kimwipes to cover the work area When done using the PCR Clean Work Station after Section IV G wipe the working area dry with kimwipes and clean with 70 EtOH Once a week clean the working area with DNA OFF Solution Clean Work Station Instructions for First Strand cDNA Synthesis Section IV E 1 Bring the lsoFreeze PCR Rack and the Flipper Rack containing the SMARTer cDNA synthesis re agents from the 20 C freezer to the PCR clean work station and put them on top of the kimwipes Take the cell or RNA samples from 80 C and put them on the IsoFreeze PCR Rack in the PCR clean work station Set up the cDNA synthesis reaction s and put the sample s in the PCR thermal cycler for cDNA synthesis Place all the used tips and tubes in a trash bag remove from the PCR clean work station and dispose Put the IsoFreeze PCR Rack and the Flipper Rack containing the SMARTer cDNA synthesis reag
33. ww clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Clontech the Clontech logo Advantage SMART SMARTer and SMARTScribe are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is aTakara Bio Company 2011 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department www clontech com Clontech Laboratories Inc ATakara Bio Company
34. your RNA is intact and free of contaminants The assay is very sensitive to variations in pipette volume etc Please make sure all your pipettes are calibrated for reliable delivery and make sure nothing is attached to the outside of the tips All lab supplies related to SMARTer cDNA synthesis need to be stored in a DNA free closed cabinet Reagents for SMARTer cDNA synthesis need to be stored in a freezer refrigerator that has not previ ously been used to store PCR amplicons Add enzymes to reaction mixtures last and thoroughly incorporate them by gently pipetting the reaction mixture up and down Do not increase or decrease the amount of enzyme added or the concentration of DNA in the reac tions The amounts and concentrations have been carefully optimized for the SMARTer amplification reagents and protocol If you are using this protocol for the first time we strongly recommend that you perform negative amp positive control reactions to verify that kit components are working properly www clontech com Protocol No PT5163 1 Version No PROX3693 7 SMARTer Ultra Low RNA Kit for Illumina Sequencing IV SMARTer cDNA Synthesis continued P PROTOCOL e Important Recipe Protocol No PT5163 1 Version No 8 PROX3693 C Sample Recommendations The sequence complexity and the average length of SMARTer cDNA is noticeably dependent on the quality of starting RNA material Due to the limiting sample siz
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