SMARTer® Stranded RNA
Contents
1. Cat No 634842 Not sold separately Box 1 96 ul 5 ul Box 2 48 ul 192 ul SMARTer Stranded Oligo 12 uM Control Mouse Liver Total RNA 1 ug ul SMART Stranded N6 Primer 12 uM 5X First Strand Buffer RNase Free 96 ul dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM 48 ul Dithiothreitol DTT 100 mM 96 ul SMARTScribe Reverse Transcriptase 100 U ul 2 1 Nuclease Free Water 55 ul RNase Inhibitor 40 U ul 960 ul Stranded Elution Buffer Clontech proprietary sequences 48rxns Illumina Indexing Primer Set Cat No 634845 48 ul Universal Forward PCR Primer 12 5 uM 48 ul Reverse PCR Primer Index 1 12 5 uM 48 ul Reverse PCR Primer Index 2 12 5 uM 48 ul Reverse PCR Primer Index 12 5 48 ul Reverse PCR Primer Index 4 12 5 uM 48 ul Reverse PCR Primer Index 5 12 5 uM 48 ul Reverse PCR Primer Index 6 12 5 uM 48 ul Reverse PCR Primer Index 7 12 5 48 ul Reverse PCR Primer Index 8 12 5 uM 48 ul Reverse PCR Primer Index 9 12 5 uM 48 ul Reverse PCR Primer Index 10 12 5 uM 48 ul Reverse PCR Primer Index 11 12 5 uM 48 ul Reverse PCR Primer Index 12 12 5 uM e 2x50rxns SeqAmp DNA Polymerase Cat No 638504 50 ul SeqAmp DNA Polymerase 1 25 ml SegAmp PCR Buffer 2X 080213 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 7 of 19 SMARTer Stranded RNA Seq Kit User Manual SMARTer Stranded RNA Seq Kit 96 rxns Cat No 634839 e 96rxns SMARTer Stranded2. 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 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 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 registered trademark of Illumina Inc Clontech the Clontech logo SegAmp 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 2013 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department 080213 www clontech com Page 19 of 19 Clontech Laboratories Inc A Takara Bio Company
3. 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 disturbing 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 be
4. 7 uem MIN NEN RT HER eee F T T T T T T iL H 20 35 100 150 200 300 400 500 600 1000 2000 10380 bp TET T T T T T T ipe re re 35 100 150 200 300 400 500 600 1000 2000 10380 bp Figure 3 Electropherogram 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 080213 www clontech com Page 16 of 19 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 1 100 ng 1 ul SMART Stranded N6 Primer 12 uM 0 7 ul Nuclease Free Water 9 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 combi
5. RNA Seq Kit Cat No 634843 Not sold separately Box 1 192 ul 5 ul Box 2 96 ul 384 ul 192 ul 96 ul 192 ul 3x1ml 55 ul 2 ml SMARTer Stranded Oligo 12 uM Control Mouse Liver Total RNA 1 ug ul SMART Stranded N6 Primer 12 uM 5X First Strand Buffer RNase Free dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U ul Nuclease Free Water RNase Inhibitor 40 U ul Stranded Elution Buffer Clontech proprietary sequences 2x 48rxns Illumina Indexing Primer Set Cat No 634845 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul 48 ul Universal Forward PCR Primer 12 5 uM Reverse PCR Primer Index 1 12 5 uM Reverse PCR Primer Index 2 12 5 uM Reverse PCR Primer Index 3 12 5 uM Reverse PCR Primer Index 4 12 5 uM Reverse PCR Primer Index 5 12 5 uM Reverse PCR Primer Index 6 12 5 uM Reverse PCR Primer Index 7 12 5 uM Reverse PCR Primer Index 8 12 5 uM Reverse PCR Primer Index 9 12 5 uM Reverse PCR Primer Index 10 12 5 uM Reverse PCR Primer Index 11 12 5 uM Reverse PCR Primer Index 12 12 5 uM e 2x50rxns SeqAmp DNA Polymerase Cat No 638504 50 ul 1 25 ml SegAmp DNA Polymerase SegAmp PCR Buffer 2X Storage Conditions 080213 Store Control Mouse Liver Total RNA and SMARTer Stranded Oligo at 70 C Store Stranded Elution Buffer at 20 C Once thawed the buffer c
6. 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 MagnaBlot 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 E ud MT Bow 2 _ EJk y du da be d per P 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 080213 www clontech com Page 18 of 19 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
7. 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 prior to cDNA synthesis with the SMARTer Stranded RNA Seq Kit If your sample has not been depleted of rRNA you may not obtain sufficient reads for analysis and any results you do obtain may be compromised 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 You may need to adjust the fragmentation times for RNA of intermediate lengths e 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 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 accommoda
8. 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 e Transfer the supernatant to a fresh 0 2 ml tube www clontech com Page 13 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual C PROTOCOL RNA Seq Library Amplification by PCR The purified first strand cDNA is amplified into RNA Seq Libraries using SeqAmp 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 1 16 10 12 100 9 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 1 ul 1 ul 1 ul 22 ul 2X SeqAmp PCR Buffer Universal Forward PCR Primer 12 5 uM Reverse PCR Primer 12 5 uM SegAmp DNA Polymerase Nuclease Free Water 50 ul Total volume per reaction Your select
9. Clontech Laboratories Inc SMARTer Stranded RNA Seqg Kit User Manual Cat Nos 634836 634837 634838 634839 080213 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 MM ocu PEE 3 IL Last of Comrpotients 5 HI Additional Materials Required uu eicere rent terrre 9 IV General Considerations u R 10 A Recommendations for Preventing eene ener enini nre 10 B General Requirements erae Certe 10 C Sample Preparation iter erre GE ER 11 D Sample Requireiierits uuu sasatan a E AEE A AE E AA Aak 11 E Sequencing Analysis Guidelines eese enne nnne inrer innen reset nennen nnns 11 Ve Protocols a q nu nana RE E OE EEEE EE EE EE RAE 12 A PROTOCOL First Strand cDNA Synthesis eese eee eee nnne erre 12 B PROTOCOL Purification of First Strand cDNA using SPRI AMPure Beadis eee 13 C PROTOCOL RNA Seq Library Amplification by PCR essere eerte 14 D PROTOCOL Purification of the RNA Seq Library using SPRI AMPure Bead
10. ads 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 for 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 15 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual E PROTOCOL Validation Using the Agilent 2100 Bio Analyzer 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 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
11. an be stored at Room Temperature Store all other reagents at 20 C www clontech com Clontech Laboratories Inc A Takara Bio Company Page 8 of 19 SMARTer Stranded RNA Seq Kit User Manual Ill 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 Filter pipette tips 10 ul 20 ul and 200 one box each e OneQuickSpin minicentrifuge for 0 2 ml tubes For PCR Amplification amp Validation e 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 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 080213 www clontech com Page 9 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stra
12. ately Box 1 241 SMARTer Stranded Oligo 12 uM 5 ul Control Mouse Liver Total RNA 1 ug ul Box 2 12 ul SMART Stranded Primer 12 uM 48 ul 5X First Strand Buffer RNase Free 24 ul dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM 12 ul Dithiothreitol DTT 100 mM 24 ul SMARTScribe Reverse Transcriptase 100 U l 1 ml Nuclease Free Water 55 ul RNase Inhibitor 40 240 ul Stranded Elution Buffer Clontech proprietary sequences 12rxns Illumina Indexing Primer Set Cat No 634844 12 ul Universal Forward PCR Primer 12 5 uM 12 ul Reverse PCR Primer Index 1 12 5 uM 12 ul Reverse PCR Primer Index 2 12 5 uM 12 ul Reverse PCR Primer Index 12 5 uM 12 ul Reverse PCR Primer Index 4 12 5 uM 12 ul Reverse PCR Primer Index 5 12 5 uM 12 ul Reverse PCR Primer Index 6 12 5 uM 12 ul Reverse PCR Primer Index 7 12 5 uM 12 ul Reverse PCR Primer Index 8 12 5 uM 12 ul Reverse PCR Primer Index 9 12 5 12 ul Reverse PCR Primer Index 10 12 5 uM 12 ul Reverse PCR Primer Index 11 12 5 uM 12 ul Reverse PCR Primer Index 12 12 5 uM e 50rxns SeqAmp DNA Polymerase Cat No 638504 50 ul SeqAmp DNA Polymerase 1 25 ml SegAmp PCR Buffer 2X 080213 www clontech com Page 5 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual SMARTer Stranded RNA Seq Kit 24 rxns Cat No 634837 e 24rxns SMARTer Stranded RNA Seq Kit Cat No 634841 Not sold separately Bo
13. indexing primer Amplify cDNA by PCR with Illumina Indexing Primer Set Read 1 gt RNA Seq library umm mm m L T Read 2 Figure 2 Flowchart of SMARTer Stranded RNA Seq library generation 080213 www clontech com Page 4 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual ll List of Components 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 The SMARTer Stranded RNA Seq Kits Cat Nos 634836 634837 634838 amp 634839 consist of e The SMARTer Stranded RNA Seq Components Cat No 634840 634841 634842 or 634843 e Illumina Indexing Primer Set Cat No 634844 or 634845 which contains PCR primers for the amplification of indexed paired end Illumina compatible sequencing libraries e SeqAmp DNA Polymerase Cat No 638504 a high fidelity hot start PCR enzyme that is well suited for use with the SMARTer Stranded RNA Seq Kit for NGS This optimized PCR enzyme has been shown to perform well even with challenging templates containing GC rich and AT rich regions The specific composition of each kit is as follows SMARTer Stranded RNA Seq Kit 12 rxns Cat No 634836 e 12rxns SMARTer Stranded RNA Seq Kit Cat No 634840 Not sold separ
14. ion 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 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 o 98 C 15 sec 55 C 15 sec 68 C 30 sec 4 forever The number of cycles depends on the amount of input RNA See Table 1 above for guidelines 080213 www clontech com Page 14 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual D PROTOCOL Purification of the RNA Seq Library using SPRI ANPure 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 080213 l 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 ofthe wall Place the sample tubes on the Magnetic Separation Device for
15. les in Appendix A instead because additional fragmentation is unnecessary and will result in lower library yields Mix the following components on ice 1 8 ul RNA 1 100 ng 1 ul SMART Stranded 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 5 min then place 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 Prepare enough Master Mix for all reactions plus 10 by combining the following reagents in the order shown on ice 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 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 Terminate the reacti
16. ludes 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 kit consists of the SMARTer Stranded RNA Seq Components SeqAmp M DNA Polymerase and the Indexing Primer Set 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 Kit utilizes our patented SMART Switching Mechanism At 5 end of RNA Transcript technology coupled with PCR amplification to generate Illumina compatible libraries without the need for 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 Start with polyA purified or ribosmal RNA depleted RNA Section IV C i SMARTer First strand cDNA Synthesis 1hr 45 min Section V A SMARTer First strand cDNA Purification Section V B RNA Seq Library Amplification by PCR 50 min Section V C RNA Seq Library Purification Section V D 20 min RNA Seq Library Validation 45 60 Section V E min Figure 1 SMARTer Stranded RNA Seq Kit protocol overview You can complete
17. lume 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 yl of freshly made 8096 ethanol to each sample without disturbing the beads in order to wash away contaminants 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 NOTE Under or over drying the beads will reduce PCR efficiency resulting in lower yields If using more than 12 PCR cycles 10 ng input RNA elute the cDNA in 20 ul of Nuclease Free Water transfer to a fresh 0 2 ml tube and repeat Steps 1 8 to ensure complete removal of adapter dimers Otherwise proceed immediately to Section V C e Add 20 ul Nuclease Free Water to the pellet e Thoroughly resuspend the beads and allow to rehydrate for 2 min e
18. nded RNA Seq Kit User Manual IV 080213 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 APCR 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 Asecond 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 separate PCR machine inside the PCR workstation for cDNA 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
19. ning 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 Transcriptase 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 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 5 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 080213 www clontech com Page 17 of 19 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
20. on 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 www clontech com Page 12 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 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 080213 B by a magnetic separation The beads are then directly used for RNA Seq library amplification NOTES Aliquot SPRI beads and allow them to come to room temperature for 30 min prior to use Before use beads should be brought to room temperature and mixed well to disperse 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 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 100 bp cDNA fragments follow this procedure for each reaction tube l m 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 vo
21. 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 10 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual 080213 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
22. s 15 E PROTOCOL Validation Using the Agilent 2100 BioAnalyzer na eene nenne 16 RAM TRE 16 Appendix A First Strand cDNA Synthesis Protocol for Degraded Samples eene 17 Appendix B Constructing a Magnetic Separation Device for 0 2 ml PCR Tubes esee 18 Table of Figures Figure 1 SMARTer Stranded RNA Seq Kit protocol overview n nennen nennen ener innen 3 Figure 2 Flowchart of SMARTer Stranded RNA Seq library generation nn 4 Figure 3 Electropherogram example results from Agilent 2100 bioanalyzer eese 16 Figure 4 Setup for positioning 0 2 ml tubes containing first strand cDNA on a MagnaBlot II Magnetic Separator 18 Figure 5 Constructing a magnetic separation device for 0 2 ml tubes from rare earth magnets 18 Table of Tables Table 1 Cycling Guidelines Based on Amount of Starting Material esee ener ener nnns 14 080213 www clontech com Page 2 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual l Introduction SMARTer cDNA Synthesis for the Sequencing Platform The SMARTer Stranded RNA Seq Kit Cat Nos 634836 634837 634838 amp 634839 inc
23. tes up to 8 of input RNA This protocol has been optimized for cDNA synthesis starting from 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 11 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual V 080213 Protocols PROTOCOL First 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 A 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 bp For some RNA samples or sequencing applications it may be appropriate to titrate the fragmentation time to achieve optimal yield and library size When working with degraded RNA samples such as FFPE RNA use the First Strand cDNA Synthesis Protocol for Degraded Samp
24. this protocol in less than 4 hr 080213 www clontech com Page 3 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Stranded RNA Seq Kit User Manual The SMARTer Stranded RNA Seq Kit starts with less than nanogram amounts of RNA A modified N6 primer the SMART Stranded N 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 see Appendix A for a fragmentation free protocol When 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 carefully designed SMARTer Stranded Oligo 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 Stranded Oligo RNA 00 3 2 SMART Stranded SMARTer Stranded N6 Primer Oligo First strand synthesis and tailing by RT am SNAARAARARAAARARAP NENNEN 1 yo 5 Template switching and extension by RT Universal Forward PCR Primer 9 V VVVVVVVVVVVVVVV N 5 X em XX m Reverse PCR
25. x 1 48 ul 5 ul Box 2 24 ul 96 ul 48 ul 24 ul 48 ul 1 ml 55 ul 480 ul SMARTer Stranded Oligo 12 uM Control Mouse Liver Total RNA 1 ug ul SMART Stranded N6 Primer 12 uM 5X First Strand Buffer RNase Free dNTP Mix dATP dCTP dGTP and dTTP each at 10 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U ul Nuclease Free Water RNase Inhibitor 40 U ul Stranded Elution Buffer Clontech proprietary sequences 2x 12 Illumina Indexing Primer Set Cat No 634844 Universal Forward PCR Primer 12 5 uM 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul 12 ul Reverse PCR Primer Index 1 12 5 uM Reverse PCR Primer Index 2 12 5 uM Reverse PCR Primer Index 3 12 5 uM Reverse PCR Primer Index 4 12 5 uM Reverse PCR Primer Index 5 12 5 uM Reverse PCR Primer Index 6 12 5 uM Reverse PCR Primer Index 7 12 5 uM Reverse PCR Primer Index 8 12 5 uM Reverse PCR Primer Index 9 12 5 uM Reverse PCR Primer Index 10 12 5 uM Reverse PCR Primer Index 11 12 5 uM Reverse PCR Primer Index 12 12 5 uM e 50rxns SeqAmp DNA Polymerase Cat No 638504 50 ul 1 25 ml 080213 SeqAmp DNA Polymerase SegAmp PCR Buffer 2X www clontech com Clontech Laboratories Inc A Takara Bio Company Page 6 of 19 SMARTer Stranded RNA Seq Kit User Manual SMARTer Stranded RNA Seq Kit 48 rxns Cat No 634838 e 48rxns SMARTer Stranded RNA Seq Kit
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