SMARTer® Stranded RNA
Contents
1. 20 Figure 5 Constructing a magnetic separation device for 0 2 ml tubes from rare earth Magnets eee aaa aaa 20 Table of Tables Table 1 Cycling Guidelines Based on Amount of Starting Material 0 0 eee kasa kasikais aaa aaa aa aaa aaa aaa 15 050714 www clontech com Page 2 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 050714 Introduction SMARTer cDNA Synthesis for the Illumina Sequencing Platform The SMARTer Stranded RNA Seq Kit Cat Nos 634836 634837 634838 634839 amp 634861 and the SMARTer Stranded RNA Seq Kit HT Cat No 634862 include the components needed to generate indexed cDNA libraries suitable for next generation sequencing NGS on any Illumina platform starting from as little as 100 pg of polyA purified or ribosomal RNA depleted RNA The kits consist of the SMARTer Stranded RNA Seq Components Se AmpTM DNA Polymerase and the Illumina Indexing Primer Set or the Indexing Primer Seq HT for Illumina PCR primers for the amplification of indexed paired end Illumina compatible sequencing libraries which enable multiplexing of NGS library analysis The entire library construction protocol can be completed in less than 4 hr Figure 1 The SMARTer Stranded RNA Seq Kits utilizes our patented SMART Switching Mechanism At 5 end of RNA Template technology coupled with PCR amplification to generate Illumina compatible libraries without the need fo2. Clontech proprietary sequences www clontech com Page 8 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Se Kit User Manual Indexing Primer Set HT for Illumina Not sold separately Store at 20 C Forward PCR Primer HT Index 1 F1 12 5 uM 15 ul Forward PCR Primer HT Index 2 F2 12 5 uM 15 ul Forward PCR Primer HT Index 3 F3 12 5 uM 15 ul Forward PCR Primer HT Index 4 F4 12 5 uM 15 ul Forward PCR Primer HT Index 5 F5 12 5 uM 15 ul Forward PCR Primer HT Index 6 F6 12 5 uM 15 ul Forward PCR Primer HT Index 7 F7 12 5 uM 15 ul Forward PCR Primer HT Index 8 F8 12 5 uM 15 ul Reverse PCR Primer HT Index 1 R1 12 5 uM 12 ul Reverse PCR Primer HT Index 2 R2 12 5 uM 12 ul Reverse PCR Primer HT Index 3 R3 12 5 uM 12 ul Reverse PCR Primer HT Index 4 R4 12 5 uM 12 ul Reverse PCR Primer HT Index 5 R5 12 5 uM 12 ul Reverse PCR Primer HT Index 6 R6 12 5 uM 12 ul Reverse PCR Primer HT Index 7 R7 12 5 uM 12 ul Reverse PCR Primer HT Index 8 R8 12 5 uM 12 ul Reverse PCR Primer HT Index 9 R9 12 5 uM 12 ul Reverse PCR Primer HT Index 10 R10 12 5 uM 12 ul Reverse PCR Primer HT Index 11 R11 12 5 uM 12 ul Reverse PCR Primer HT Index 12 R12 12 5 uM 12 ul Indexing Primer Set HT for Illumina sequences Index Barcode Index Barcode tube label tube label F1 TATAGCCT R1 ATTACTCG F2 ATAGAGGC R2 TCCGGAGA F3 CCT
3. provided in Section V A e After RNA extraction if your sample amount is not limiting we recommend evaluating total RNA quality using the Agilent RNA 6000 Pico Kit Cat No 5067 1513 Input RNA purity and quantity The input amounts indicated in this kit are for poly A purified rRNA depleted or otherwise purified RNA samples e Purity of input RNA Input RNA should be free from genomic or carrier DNA and contaminants that would interfere with oligo annealing or reverse transcriptase reactions IMPORTANT Purified total RNA should be resuspended in nuclease free water not in TE or other buffers containing EDTA Chelation of divalent cations by EDTA will interfere with RNA fragmentation e Volume and amount of input RNA This kit accommodates up to 8 ul of input RNA This protocol has been optimized for cDNA synthesis starting from 0 1 ng of RNA However if your RNA sample is not limiting we recommend that you start with more than 1 ng of RNA Sequencing Analysis Guidelines e The first three bases of the first sequencing read Read 1 are derived from the SMARTer Stranded Oligo We recommend trimming these bases prior to mapping e Read 1 is derived from the sense strand of the input RNA If you are performing paired end sequencing Read 2 corresponds to the antisense strand www clontech com Page 12 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual V Protocols A PROTOCOL Firs
4. separation device for 0 2 ml tubes see Appendix B NOTE We strongly recommend using separate magnets for purification of first strand cDNA Section V B and purification of the RNA seq library Section V D to prevent cross contamination e 80 ethanol 050714 www clontech com Page 10 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual IV 050714 General Considerations A Recommendations for Preventing Contamination 1 Before you set up the experiment it is advisable to have two physically separated work stations e A PCR Clean Work Station for all pre PCR experiments that require clean room conditions such as first strand cDNA synthesis Section V A and purification of first strand cDNA Section V B e A second work station located in the general laboratory where you will perform PCR to amplify the RNA seq library Section V C purify the RNA seq library Section V D and measure its concentration Section V E IMPORTANT The PCR work station must be located in a clean room with positive air flow as contamination may occur very easily Once contamination occurs it can be difficult to remove Guidelines for clean room operation e Only move materials supplies 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 e Use a separate PCR machine inside the PCR workstation for cD
5. 1 GGCTAC GCCAAT 12 CTTGTA www clontech com Page 7 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Se Kit User Manual 050714 The SMARTer Stranded RNA Seq Kit HT consists of the SMARTer Stranded RNA Seq Components not sold separately the Indexing Primer Set HT for Illumina not sold separately and Se Amp DNA Polymerase These 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 SMARTer Stranded RNA Seq Kit HT 2 SeqAmp DNA Polymerase Store at 20 C SegAmp DNA Polymerase 2x 50 ul SeqAmp PCR Buffer 2x 2x 1 25 ml SMARTer Stranded RNA Seq Components Not sold separately Package 1 Store at 70 C SMARTer Stranded Oligonucleotide 12 uM 192 ul Control Mouse Liver Total RNA 1 ug ul 5 ul Package 2 Store at 20 C Once thawed store Stranded Elution Buffer at Room Temperature Continue to store all other reagents at 20 C SMARTer Stranded N6 Primer 12 uM 96 ul 5X First Strand Buffer RNase Free 384 ul dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM 192 ul Dithiothreitol DTT 100 mM 96 ul SMARTScribe Reverse Transcriptase 100 U l 192 ul Nuclease Free Water 3x1ml RNase Inhibitor 40 U ul 55 ul Stranded Elution Buffer 2ml
6. ARTer Stranded RNA Seq Kit User Manual 050714 C E Sample Preparation The sequence complexity and the average length of SMARTer cDNA is noticeably dependent on the quality of starting RNA material e 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 e When choosing a purification method kit ensure that it is appropriate for your sample amount Sample Requirements Ribosomal RNA rRNA depletion We strongly recommend removing rRNA from the sample prior to cDNA synthesis using the SMARTer Stranded RNA Seq Kit For 10 100 ng of input total RNA we recommend the RiboGone Mammalian Cat Nos 634846 and 634847 rRNA depletion kit This kit specifically and efficiently degrades 5S 5 65 18S and 28S nuclear rRNA and 72S mtRNA from mouse rat and human total RNA samples For 2 10 ng of input total RNA we recommend using a modified protocol with Ribo Zero technology from Illumina To find the protocol go to www clontech com and type Protocol for Removal of rRNA from Small Amounts of Total RNA in the search box Input RNA length e The SMARTer Stranded RNA Seq Kit was developed for full length intact RNA An alternative protocol for degraded samples is available in Appendix A Fragmentation times for RNA of intermediate lengths are
7. ATCCT R3 CGCTCATT F4 GGCTCTGA R4 GAGATTCC F5 AGGCGAAG R5 ATTCAGAA F6 TAATCTTA R6 GAATTCGT F7 CAGGACGT R7 CTGAAGCT F8 GTACTGAC R8 TAATGCGC R9 CGGCTATG R10 TCCGCGAA R11 TCTCGCGC R12 AGCGATAG 050714 www clontech com Page 9 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual lll 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 e Single channel pipette 10 ul 20 ul and 200 ul one each e Eight channel pipette 10 ul for the SMARTer Stranded RNA Seq Kit HT only e Twelve channel pipette 10 ul for the SMARTer Stranded RNA Seq Kit HT only e Filter pipette tips 10 ul 20 ul and 200 ul one box each and two additional boxes of 10 ul tips for the SMARTer Stranded RNA Seq Kit HT e One QuickSpin minicentrifuge for 0 2 ml tubes For PCR Amplification amp Validation e One dedicated PCR thermal cycler used only for first strand synthesis e High Sensitivity DNA Kit Agilent Cat No 5067 4626 e Nuclease free thin wall PCR tubes 0 2 ml USA Scientific Cat No 1402 4700 e Nuclease free nonsticky 1 5 ml tubes USA Scientific Cat No 1415 2600 For SPRI Bead Purification e Agencourt AMPure PCR Purification Kit 5 ml Beckman Coulter Part No A63880 60 ml Beckman Coulter Part No A63881 e Magnetic
8. Clontech Laboratories Inc SMARTer Stranded RNA Seg Kit User Manual Cat Nos 634836 634837 634838 634839 634861 634862 050714 Clontech Laboratories Inc A Takara Bio Company 1290 Terra Bella Avenue Mountain View CA 94043 USA U S Technical Support tech clontech com United States Canada Asia Pacific Europe Japan 800 662 2566 1 650 919 7300 33 0 1 3904 6880 81 0 77 543 6116 SMARTer Stranded RNA Seq Kit User Manual Table of Contents I MPPO GUC HOM ass ia ri i gece sete i lee dee D i wed a e us teen Delt i i a i i i a i a a i a EE E 3 I Listof Components ia sasisiin ss Cex ai aisiais i aa i E i ai ia a a a ni ES 6 III Additional Materials REQUITEG is ccossseisceslenscasssteseanveadesen ss K o a ia a ss i i a so ESEESE 10 IV General ConsideratiONS Lisnis stisis iki spiabs iskelia I e i i i i a a i a a 11 A Recommendations for Preventing Contamination saaa aaa aaa aa aka aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa 11 By General Requirements 5i ssi csdccuoss seis tecesesuve Dik i o o S paso L A a a k a wees 11 C Sample Preparation Lei is sai iii a i i i a a g i so k i k i aeaiia 12 D Sample Requirement ss csc sices5e tyocs tataigen eeen a Ea lean a Selted EE E AE idinigens Pel oat nego E EE 12 E Seg uencing Analysis Guidelines sstsisii ses aion esi ais i oeaio d EN i a a i i i i a a 12 Ve Protocols skitais ives La ki tani ia is L a i i a E Gib i i i a a i D a sai a Las 13 A PROTOCOL F
9. NA synthesis e Wear gloves and sleeve covers throughout the procedure to protect your RNA samples from degradation 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 RNA sample Prior to cDNA synthesis please make sure that your RNA is free of contaminants The assay is very sensitive to variations in pipette volume etc Please make sure that all your pipettes are calibrated for reliable delivery and that 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 previously 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 reactions 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 and positive control reactions to verify that kit components are working properly www clontech com Page 11 of 21 Clontech Laboratories Inc A Takara Bio Company SM
10. agnaBlot II Magnetic Separator Promega Part No V8351 Figure 4 Setup for positioning 0 2 ml tubes containing first strand cDNA on a MagnaBlot II Magnetic Separator Example 2 Building a 0 2ml tube magnetic separation device from rare earth bar magnets and a tip rack As seen in Figure 5 neodymium bar magnets are taped together on the underside of the top section of a 20 ul tip rack Panel A and the rack is inverted so the tubes can be inserted Panel B G gt ea ey ey T N pe JGH be Ja ton hh Figure 5 Constructing a magnetic separation device for 0 2 ml tubes from rare earth magnets Panel A shows six 0 75 x 0 25 x 0 5 neodymium bar magnets Applied Magnets Model NB026 taped together on the underside of the top section of a 20 ul tip rack Panel B shows the upright rack into which an 8 tube strip of 0 2 ml tubes has been inserted 050714 www clontech com Page 20 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 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 Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose includ
11. beads are then directly used for RNA seq library amplification NOTES e Aliquot SPRI beads and allow them to come to room temperature for 30 min prior to use e Before use beads should be brought to room temperature and mixed well to disperse e You will need a Magnetic Separation Device for 0 2 ml tubes If you do not have such a device we recommend constructing one using the instructions in Appendix B e Clean up of SMARTer reactions must be performed using Ampure XP beads Spin columns do not adequately remove adapter dimers from the reactions and will result in experimental failure To purify the SMART cDNA from unincorporated nucleotides and small lt 100 bp cDNA fragments follow this procedure for each reaction tube 1 N 050714 Add 20 ul of SPRI AMPure beads to each sample using a 20 ul pipetter e Mix by vortexing for 5 sec or by pipetting the entire volume up and down at least 10 times e The beads are viscous suck the entire volume up and push it out slowly Incubate at room temperature for 8 min to let DNA bind to the beads Briefly spin the sample tubes to collect the liquid from the walls of the tube Place the sample tubes on the Magnetic Separation Device for 5 min or longer until the solution is completely clear While the tubes are sitting on the magnetic stand pipette out the supernatant Add 200 ul of freshly made 80 ethanol to each sample without disturbing the beads in order to wash away contam
12. eqAmp DNA Polymerase the Universal Forward PCR Primer and the Reverse PCR Primers from the Illumina Indexing Primer Set 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 Table 1 Cycling Guidelines Based on Amount of Starting Material Amount of Typical Number Input RNA ng of PCR Cycles 0 1 18 1 16 10 12 100 9 NOTE If you are using the SMARTer Stranded RNA Seq Kit HT Cat No 634862 use the alternative protocol below 1 Prepare a PCR Master Mix for all reactions Separate master mixes should be prepared for different library indexes Combine the following reagents in the order shown then mix well and spin the tube briefly in a microcentrifuge 25 ul 2X SegAmp PCR Buffer 1 ul Universal Forward PCR Primer 12 5 uM 1ul Reverse PCR Primer 12 5 uM 1 ul SegAmp DNA Polymerase 22 ul 50 ul Nuclease Free Water Total volume per reaction Your selection of the Reverse PCR Primer will determine which of the 12 indexing sequences in the Illumina Indexing Primer Set will be associated with your library 2 Add 50 ul of PCR Master Mix to each tube containing DNA bound to the beads from Section V B Step 8 Mix well making sure that the beads are uniformly resuspended 050714 www clontech com Page 15 of 21 Clontech Laboratori
13. es Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 3 Place the tube in a preheated thermal cycler with a heated lid Start thermal cycling using the following program 94 C 1 min X cycles 98 C 15sec 55 C 15 sec 68 C 30 sec 4 C forever a The number of cycles depends on the amount of input RNA See Table 1 above for guidelines For the SMARTer Stranded RNA Seq Kit HT The purified first strand cDNA is amplified into 96 uniquely indexed RNA seq libraries using Se Amp DNA Polymerase and the Forward and Reverse PCR Primers from the Indexing Primer Set HT for Illumina 1 Prepare a PCR Master Mix for all reactions Combine the following reagents in the order shown then mix well and spin the tube briefly in a microcentrifuge 25 ul 2X SeqAmp PCR Buffer 1 ul SegAmp DNA Polymerase 22 ul Nuclease Free Water 48 ul Total volume per reaction 2 Add 48 pl of PCR Master Mix to each well containing DNA bound to the beads from Section V B Step 8 Mix well making sure that the beads are uniformly resuspended 3 Using an 8 channel pipette add 1 ul of each Forward PCR Primer HT to the wells of the 96 well plate containing DNA bound to beads and the Master Mix being careful not to cross contaminate each well Each well in a row will have the same Forward PCR Primer HT Index 4 Using a 12 channel pipette add 1 ul of each Reverse PCR Primer HT to the wells of the 96 well plate being careful not to cros
14. inants Wait for 30 sec and carefully pipette out the supernatant Repeat Step 5 Perform a brief spin of the tubes 2 000 g to collect the remaining ethanol at the bottom of each tube Place the tubes on the magnetic stand for 30 sec then remove all the remaining ethanol with a pipette Let the sample tubes rest open at room temperature for 3 5 min until the pellet appears dry You may see a tiny crack in the pellet If using more than 10 ng of input RNA proceed immediately to Section V C NOTE Under or over drying the beads will reduce PCR efficiency resulting in lower yields www clontech com Page 14 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 9 If using less than 10 ng of input RNA elute the cDNA in 20 ul of Nuclease Free Water as described below step 9a d this ensures complete removal of adapter primers Then repeat steps 1 8 above before proceeding to Section V C a Add 20 ul Nuclease Free Water to the pellet from step 8 b Thoroughly resuspend the beads and allow to rehydrate for 2 min c Briefly spin the sample tubes to collect the liquid from the walls of the tube Place the sample tubes on the Magnetic Separation Device for 1 min or longer until the solution is completely clear d Transfer the supernatant to a fresh 0 2 ml tube C PROTOCOL RNA Seq Library Amplification by PCR The purified first strand cDNA is amplified into RNA seq libraries using S
15. ing 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 prior written approval of Clontech Laboratories Inc Your use of this product is also subject to compliance with any applicable licensing requirements described on the product s web page at http www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Illumina is a trademark of Illumina Inc Ribo Zero is a trademark of Epicentre an Illumina Company Clontech the Clontech logo RiboGone SeqAmp 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 a Takara Bio Company 2014 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department 050714 www clontech com Page 21 of 21 Clontech Laboratories Inc A Takara Bio Company
16. irst Strand CDNA Synthesis aa aa aaa aaa aaa aaa aa aa aa aa aaa aaa aaa aaa aaa aaa aaa 13 B PROTOCOL Purification of First Strand cDNA using SPRI AMPure Beads 0 ee aaa aaa aaa 14 C PROTOCOL RNA Seg Library Amplification by PCR 0 cc cecceeceseeseceseeeeseeeseeeseeeseecsaecaaecsaecsseeeseeseeesseeseneeenes 15 D PROTOCOL Purification of the RNA Seq Library using SPRI AMPure Beads 0 0 0 0 ceeeesceseceseceeeeeeeeeeneeenee 17 E PROTOCOL Validation Using the Agilent 2100 BioanalyZer oe aa aa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa 18 VI REIEIENC S 52 tibia as Sai ii a a aa cage a ii i a i a a a ia i a e a a 18 Appendix A First Strand cDNA Synthesis Protocol for Degraded Samples 0 eeeeceesceeseeeseeeseeesceeseeesaeceseceaeeeaeeeseeess 19 Appendix B Constructing a Magnetic Separation Device for 0 2 ml PCR Tube 00 cece eeeeeseeeseeesceeneeenaeceseenaeseaeeeseeees 20 Table of Figures Figure 1 SMARTer Stranded RNA Seq Kit protocol overview 2 0 eeceescessseessecsseeeseceseeeseceseeeeeeeeneeeaeeeaeeeaeecaaecsaecaeseaeenseenes 4 Figure 2 Flowchart of SMAR Ter Stranded RNA Seq Kit library generation 0 0 ee eee eseeseeeneeeneeceaeceseceseeeeeeseeeseeeeneeeaee 5 Figure 3 Electropherogram example results from Agilent 2100 BioanalyZet eee eeeeeeseseeeneeereeeeeeseeesaeeeseceaeeeseenseeees 18 Figure 4 Setup for positioning 0 2 ml tubes containing first strand cDNA on a MagnaBlot II Magnetic Separator
17. isturbing the beads to wash away contaminants Wait for 30 sec and carefully pipette out the supernatant DNA will remain bound to the beads during the washing process Repeat Step 5 one more time Perform a brief spin of the tubes 2 000 g to collect the remaining ethanol at the bottom of each tube Place the tubes on the magnetic stand for 30 sec then remove all the remaining ethanol with a pipette Let the sample tubes rest open at room temperature for 3 5 min until the pellet appears dry You may see a tiny crack in the pellet NOTE Be sure to dry the pellet enough e If you under dry the pellet ethanol will remain in the sample tubes The ethanol will reduce your RNA seq library recovery rate and ultimately your yield Allow the tubes to sit at room temperature until the pellet is dry e If you over dry the pellet it will take longer than 2 min to rehydrate Step V D 9 Once the beads are dried add 20 ul of Stranded Elution Buffer to cover the beads Remove the tubes from the magnetic stand and mix thoroughly to resuspend the beads NOTE Be sure the beads are completely resuspended The beads can sometimes stick to the sides of the tube We recommend vortexing or directly pipetting the beads up and down to ensure complete dispersion Incubate at room temperature for 2 min to rehydrate Briefly spin the sample tubes to collect the liquid from the side of the wall Place the sample tubes on the Magnetic Separation Device fo
18. pherogram example results from Agilent 2100 Bioanalyzer VI References Chenchik A et al 1998 In RT PCR Methods for Gene Cloning and Analysis BioTechniques Books MA pp 305 319 050714 www clontech com Page 18 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual Appendix A First Strand cDNA Synthesis Protocol for Degraded Samples Our typical protocol for first strand cDNA synthesis Section V A includes simultaneous RNA fragmentation If your RNA is already fragmented use this alternative protocol for first strand cDNA synthesis 1 Mix the following components on ice 1 8 ul RNA 0 1 100 ng 1 ul SMART Stranded N6 Primer 12 uM 0 7 ul Nuclease Free Water 9ul Total volume 2 Incubate the tubes at 72 C in a preheated hot lid thermal cycler for 3 min then put the samples on ice for 2 min NOTE Steps 4 6 should not be delayed after completing Step 2 since they are critical for first strand cDNA synthesis You can prepare your master mix except for SMARTScribe Reverse Transcriptase for Step 3 while your tubes are incubating Step 2 in order to jump start the cDNA synthesis 3 Prepare enough Master Mix for all reactions plus 10 by combining the following reagents in the order shown on ice 4ul 5X First Strand Buffer 0 5 ul DTT 100 mM 0 5 ul RNase Inhibitor 2ul dNTP Mix 10 mM 2ul SMARTer Stranded Oligo 12 uM 2 ul SMARTScribe Reverse Transcrip
19. r 1 min or longer until the solution is completely clear Transfer the clear supernatant containing the purified RNA seq library from each tube to a nuclease free nonsticky tube www clontech com Page 17 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Se Kit User Manual E PROTOCOL Validation Using the Agilent 2100 Bioanalyzer 1 Dilute 1 ul of the amplified RNA seq library with 3 ul Stranded Elution Buffer 2 Use 1 ul of the diluted sample for validation using the Agilent 2100 Bioanalyzer and the High Sensitivity DNA Chip from Agilent s High Sensitivity DNA Kit Cat No 5067 4626 See the user manual for the Agilent High Sensitivity DNA Kit for instructions 3 Compare the results for your samples and controls if performed to determine whether the sample is suitable 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 150 1 000 bp peaked at 300 bp for the positive control RNA sample Figure 3 Panel A yielding gt 7 5 nM RNA seq library depending on the input and number of cycles Positive Control RNA Negative Control ma ee oetaon ce eee ee ee EO OR ais al M A E fi Meet ii T T T T T T IE i ii 204 35 100 150 200 300 400 500 600 1000 2000 10380 bp ial T T T T T T re r 35 100 150 200 300 400 500 600 1000 2000 10380 bp Figure 3 Electro
20. r SMARTScribe Reverse Transcriptase for Step 3 while your tubes are incubating Step 2 in order to jump start the cDNA synthesis Prepare enough Master Mix for all reactions plus 10 by combining the following reagents in the order shown on ice 0 5 ul 0 5 ul 2 ul 2 ul 2 ul DTT 100 mM RNase Inhibitor dNTP Mix 10 mM SMARTer Stranded Oligo 12 uM SMARTScribe Reverse Transcriptase 100 U ul 7 ul Total volume per reaction Add the reverse transcriptase to the master mix immediately prior to use Mix well by gently vortexing and spin the tubes briefly in a microcentrifuge Add 7 ul of the Master Mix to each reaction tube from Step 2 Mix the contents of the tubes by gently pipetting and spin the tubes briefly to collect the contents at the bottom Incubate the tubes in a preheated thermal cycler at 42 C for 90 min www clontech com Page 13 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 6 Terminate the reaction by heating the tubes at 70 C for 10 min then leave them in the thermal cycler at 4 C until the next step Section V B 1 NOTE If desired you may stop here and store the reaction tubes at 4 C overnight before proceeding to Section V B B PROTOCOL Purification of First Strand cDNA using SPRI AMPure Beads The first strand cDNA selectively binds to SPRI beads leaving contaminants in solution which is removed by a magnetic separation The
21. r enzymatic clean up or adapter ligations The directionality of the template switching reaction preserves the strand orientation of the RNA making it possible to obtain strand specific sequencing data from the synthesized cDNA www clontech com Page 3 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual Start with polyA purified or ribosmal RNA depleted RNA Section IV C SMARTer First strand cDNA Synthesis 1 hr 45 min i Section V A SMARTer First strand cDNA Purification i Section V B RNA Seq Library Amplification by PCR 50 min Section V C RNA Seg Library Purification Section V D 20 min RNA Seg Library Validation 45 60 Section V E min Figure 1 SMARTer Stranded RNA Se Kit protocol overview You can complete this protocol in less than 4 hr The SMARTer Stranded RNA Seq Kits start with sub nanogram amounts of RNA A modified N6 primer the SMART Stranded N6 Primer primes the first strand synthesis reaction Figure 2 For added simplicity the RNA is chemically fragmented during denaturation NOTE If your sample is degraded or of low quality RNA Integrity Number RIN lt 3 see Appendix A for a fragmentation free protocol When the SMARTScribe Reverse Transcriptase reaches the 5 end of the RNA fragment the enzyme s terminal transferase activity adds a few additional nucleotides to the 3 end of the cDNA The ca
22. randed RNA Seq Kit consists of the SMARTer Stranded RNA Seq Components not sold separately the Illumina Indexing Primer Set not sold separately and Se Amp DNA Polymerase These 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 634836 634837 634838 634839 SMARTer Stranded RNA Se Kit 7 12rxns 24 rxns 48 rxns 96 rxns SeqAmp DNA Polymerase Store at 20 C SeqAmp DNA Polymerase 50 ul 50 ul 2x 50 ul 2 x 50 ul SeqAmp PCR Buffer 2x 1 25 ml 125ml 2x1 25ml 2x1 25ml SMARTer Stranded RNA Seq Components Not sold separately Storage conditions are listed below for Package 1 and Package 2 Package 1 Store at 70 C SMARTer Stranded Oligonucleotide 12 uM 24 ul 48 ul 96 ul 192 ul Control Mouse Liver Total RNA 1 ug ul 5 ul 5 ul 5 ul 5 ul Package 2 Store at 20 C Once thawed store Stranded Elution Buffer at Room Temperature Continue to store all other reagents at 20 C SMARTer Stranded N6 Primer 12 uM 12 ul 24 ul 48 ul 96 ul 5X First Strand Buffer RNase Free 48 ul 96 ul 192 ul 384 ul dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM 24 ul 48 ul 96 ul 192 ul Dithiothreitol DTT 100 mM 12 ul 24 ul 48 ul 96 ul SMARTScribe Reverse Transcriptase 100 24
23. refully designed SMARTer Stranded Oligo base pairs with the non templated nucleotide stretch creating an extended template to enable the SMARTScribe RT to 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 Stranded Oligo 050714 www clontech com Page 4 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Se Kit User Manual RNA ue X SNANVVYVYNYNYNYNYNYNNNNNANNS 3 5 SMART Stranded SMARTer Stranded N6 Primer Oligo First strand synthesis and tailing by RT X p PPP LPIPIPIPIIPIIIIISII wk X Template switching and extension by RT Universal Forward PCR Primer 2 uu S AAA AAAA NINDS X Reverse PCR indexing primer Amplify cDNA by PCR with Illumina Indexing Primer Set Read 1 gt RNA Seg library SSS VA SS A A lt Read2 Figure 2 Flowchart of SMARTer Stranded RNA Seq Kit library generation The SMARTer Stranded RNA Seq Kit HT follows the same method for library generation but uses eight different Forward PCR indexing primers along with the twelve Reverse PCR indexing primers to generate 96 uniquely indexed cDNA libraries 050714 www clontech com Page 5 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Se Kit User Manual 050714 List of Components The SMARTer St
24. s contaminate each well Each well in a column will have the same Reverse PCR Primer HT Index 5 Place the tube in a preheated thermal cycler with a heated lid Start thermal cycling using the following program 94 C 1 min X cycles 98 C 15 sec 55 C 15 sec 68 C 30 sec 4 C forever a The number of cycles depends on the amount of input RNA See Table 1 above for guidelines 050714 www clontech com Page 16 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual D PROTOCOL Purification of the RNA Seg Library using SPRI AMPure Beads The amplified RNA seq library is purified by immobilizing it onto SPRI beads The beads are then washed with 80 ethanol and eluted in Stranded Elution Buffer 050714 1 10 11 12 Add 50 ul of SPRI AMPure beads to each sample e Mix by vortexing 5 sec or by pipetting the entire volume up and down at least 10 times e The beads are viscous suck the entire volume up and push it out slowly Incubate at room temperature for 8 min to let the DNA bind to the beads Briefly spin the sample tubes to collect the liquid from the side of the wall Place the sample tubes on the Magnetic Separation Device for 5 min or longer until the solution is completely clear While the tubes are sitting on the magnetic stand pipette out the supernatant Keep the tubes on the magnetic stand Add 200 ul of freshly made 80 ethanol to each sample without d
25. t Strand cDNA Synthesis During this step RNA is fragmented and converted to single stranded ss cDNA that contains sequences complementary to the SMARTer Stranded Oligo IMPORTANT e The following protocol is designed for full length undegraded RNA The first two steps will simultaneously fragment and prime the RNA for cDNA synthesis NOTE This protocol was designed to fragment full length polyadenylated RNA for a final mean library insert size of 180 nt For some RNA samples or sequencing applications it may be appropriate to titrate the fragmentation time to achieve optimal yield and library size e When working with degraded RNA samples with RIN lt 3 such as FFPE RNA use the First Strand cDNA Synthesis Protocol for Degraded Samples in Appendix A instead because additional fragmentation is unnecessary and will result in lower library yields 1 Mix the following components on ice 050714 1 8 ul RNA 0 1 100 ng 1 ul SMART Stranded N6 Primer 12 uM 4ul 5X First Strand Buffer RNase Free 0 7 ul Nuclease Free Water 13 ul Total volume Incubate the tubes at 94 C in a preheated hot lid thermal cycler for an appropriate time depending on the RIN of the RNA sample then place the samples on ice for 2 min RIN Time gt 7 5 min 4 7 4min 3 3 min NOTE Steps 4 6 should not be delayed after completing Step 2 since they are critical for first strand cDNA synthesis You can prepare your master mix except fo
26. tase 100 U ul 11 ul Total volume per reaction Add the reverse transcriptase to the master mix immediately prior to use Mix well by gently vortexing and spin the tubes briefly in a microcentrifuge 4 Add 11 ul of the Master Mix to each reaction tube from Step 2 Mix the contents of the tubes by gently pipetting and spin the tubes briefly to collect the contents at the bottom Incubate the tubes in a preheated thermal cycler at 42 C for 90 minutes 6 Terminate the reaction by heating the tubes at 70 C for 10 min then leave them in the thermal cycler at 4 C until the next step Section V B 1 NOTE If desired you may stop here and store the reaction tubes at 4 C overnight before proceeding to Section V B 050714 www clontech com Page 19 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual Appendix B Constructing a Magnetic Separation Device for 0 2 ml PCR Tubes It can be difficult to find magnetic separation devices designed specifically to handle 0 2 ml PCR strip tubes Often one can place strip tubes in a column row of a magnetic separation device designed for use with 96 well plates Alternatively one can construct a suitable low cost separation device from common laboratory materials Example 1 Using a 96 well separation device with strip tubes As seen in Figure 4 you may place the tubes in the top part of an inverted P20 or P200 Rainin Tip Holder which is taped to a M
27. ul 48 ul 96 ul 192 ul U Ll Nuclease Free Water 1ml 1ml 2x1ml 3x1ml RNase Inhibitor 40 U ul 55 ul 55 ul 55 ul 55 ul Stranded Elution Buffer 240 ul 480 ul 960 ul 2ml Clontech proprietary sequences www clontech com Page 6 of 21 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Se Kit User Manual Illumina Indexing Primer Set Not sold separately Store at 20 C 050714 oahAn Universal Forward PCR Primer 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 1 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 2 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 3 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 4 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 5 12 5 uM 12 ul 2x12 ul 48 ul 2 x 48 ul Reverse PCR Primer Index 6 12 5 uM 12 ul 2x12 ul 48 ul 2x 48 ul Reverse PCR Primer Index 7 12 5 uM 12 ul 2x12 ul 48 ul 2 x 48 ul Reverse PCR Primer Index 8 12 5 uM 12 ul 2x12 ul 48 ul 2x 48 ul Reverse PCR Primer Index 9 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 10 12 5 uM 12 ul 2x12ul 48 ul 2x 48 ul Reverse PCR Primer Index 11 12 5 uM 12 ul 2x12 ul 48 ul 2 x 48 ul Reverse PCR Primer Index 12 12 5 uM 12 ul 2x12 ul 48 ul 2 x 48 ul Illumina Indexing Primer Set sequences Index Barcode Index Barcode ATCACG 7 CAGATC CGATGT 8 ACTTGA TTAGGC 9 GATCAG TGACCA 10 TAGCTT ACAGTG 1
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