SMARTer® Ultra™ Low Input RNA Kit for Sequencing
Contents
1. 14 SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Once the beads are dry add 17 ul of Elution Buffer to cover the bead pellet Remove the samples from the magnetic separation device and mix thoroughly to resuspend the beads Incubate at room temperature for 2 minutes to rehydrate Briefly spin the samples to collect the liquid from the side of the tube or sample well Place the samples back on the magnetic device for 1 minute or longer until the solution is completely clear NOTE There may be a small population of beads that do not pellet against the magnet during incubation Pipet these unpelleted beads up and down to resuspend them with the supernatant and then pipet them towards the magnet where the rest of the beads have already pelleted without disrupting the existing pellet Continue the incubation until there are no beads left in the supernatant 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 STOPPING POINT The samples may be stored at 20 C indefinitely 120514 www clontech com Page 14 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual B Validation Using the Agilent 2100 BioAnalyzer 1 Aliquot 1 ul of the amplified cDNA for validation using the Agilent 2100 BioAnalyzer and the High Sensitivity DNA Chip from Agile2. 5 minutes or longer until the liquid appears completely clear and there are no beads left in the supernatant 6 While the samples are on the magnetic separation device pipette out the supernatants 7 Keep the samples on the magnetic separation device Add 200 ul of freshly made 80 ethanol to each sample without disturbing the beads Wait for 30 seconds and carefully pipette out the supernatant containing contaminants DNA will remain bound to the beads during the washing process 8 Repeat Step 7 once 9 Briefly spin the samples to collect the liquid from the side of the wall Place the samples on the magnetic device for 30 seconds then remove all the remaining ethanol with a pipette 10 Place the samples at room temperature for approximately 2 5 3 minutes until the pellet is no longer shiny and before a crack appears NOTE Be sure to dry the pellet only until it is just dry The pellet will look matte with no shine If you under dry the pellet ethanol will remain in the sample wells The ethanol will reduce your amplified cDNA recovery rate and ultimately your yield Allow the plate to sit at room temperature until the pellet is dry If you over dry the pellet there will be cracks in the pellet It will take longer than 2 minutes to rehydrate Step V A 12 and may reduce amplified cDNA recovery and yield 120514 www clontech com Page 13 of 19 Clontech Laboratories Inc A Takara Bio Company 11 12 13
3. clontech com Page 16 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Protocol lon Torrent Sequencing Platforms Prior to generating the final library for Ion Torrent sequencing the cDNA is simultaneously digested with Afal to remove SMART adapters and enzymatically sheared using reagents from the Ion Xpress Plus Fragment Library Preparation Kit Just 1 ng of amplified cDNA is sufficient for this protocol but it is recommended that you use as much of your cDNA as possible for best results The resulting sheared cDNA from the protocol will be in the 80 600 bp size range The conditions have been optimized for library sizes yielding 200 base reads on the Ion Proton platform Shearing conditions may need to be modified for different sized libraries Refer to the Ion Xpress Plus Fragment Library Preparation User Guide for guidelines on optimizing the Ion Shear reaction time but note that the reaction time will be affected by the presence of Afal enzyme 1 Vortex the Ion Shear Plus 10X Reaction Buffer and the Ion Shear Plus Enzyme Mix II for 5 seconds each and spin briefly to collect the contents at the bottom of the tubes Store on ice Add the following reagents in the order shown to individual 0 2 ml nuclease free low adhesion tubes or an 8 well strip Mix vigorously by vortexing for 5 seconds Spin the tube s briefly in a minicentrifuge to collect the conte
4. rxns 192 rxns 480 rxns SeqAmp DNA Polymerase Store at 20 C SeqAmp DNA Polymerase 50 ul 50 ul 2x50 ul 200 ul 2 x 200 ul 3 x 200 ul SeqAmp PCR Buffer 1 25 ml 1 25 ml 2x1 25ml 4x 1 25 ml 8 x 1 25 ml 12x 1 25 ml SMARTer Ultra Low Input RNA Kit for Sequencing v3 Components Not sold separately Storage conditions are listed below for Box 1 and Box 2 Box 1 Store at 70 C SMARTer Il A Oligonucleotide 12 uM 12 ul 24 ul 48 ul 96 ul 2x 96 ul 5 x 96 ul Control Total RNA 1 5 ul 5 ul 5 ul 5 ul 2x5ul 5x5 ul gil u u u u u u Box 2 Store at 20 C Once thawed store Lysis Buffer at 4 C and store Elution Buffer at 20 C Continue to store all other reagents at 20 C 3 SMART CDS Primer ILA 12 uM 12 ul 24 ul 48 ul 96 ul 2x 96 ul 5 x 96 ul PCR Primer II A v3 12 uM 12 ul 24 ul 48 ul 96 ul 2x 96 ul 5 x 96 ul 5X First Strand Buffer 48 ul 96 ul 192 ul 384 ul 2 x 384 ul 5 x 384 ul SMARTer dNTP Mix 20 mM each 12 ul 24 ul 48 ul 96 ul 2x 96 ul 5 x 96 ul Dithiothreitol DTT 100 mM 6 ul 12 ul 24 ul 48 ul 2x 48 ul 5 x 48 ul SMARTScribe Reverse Transcriptase 24 ul 48 ul 96 ul 192 ul 2x 192 ul 5 x 192 ul 100 U ul Nuclease Free Water 2x1ml 2x1ml 2 mi 4ml 2x4ml 5x4ml RNase Inhibitor 40 U ul 30 ul 60 ul 120 ul 240 ul 2 x 240 ul 5 x 240 ul 10X Lysis Buffer v3 230 ul 460 ul 920 ul 1 85 ml 2 x 1 85 ml 5 x 1 85 ml Elution Buffer 10 mM
5. 1 ng 100 cells 12 100 pg 10 cells 15 10 pg 1 cell 18 1 Thaw all the reagents needed for PCR except the enzyme on ice Gently vortex each reagent tube to mix and spin down briefly Store on ice 2 Prepare enough PCR Master Mix for all the reactions plus 10 of the total reaction mix volume Combine the following reagents in the order shown NOTE Remove the DNA polymerase from the freezer gently mix the tube without vortexing and add to the Master Mix just before use Mix the Master Mix well by vortexing gently and spin the tube briefly in a microcentrifuge to collect the contents at the bottom of the tube 25 ul 2X SeqAmp PCR Buffer 1 ul PCR Primer Il A v3 12 uM 1 ul SeqAmp DNA Polymerase 3 ul Nuclease free water 30 ul Total Volume added per reaction 3 Add 30 ul of PCR Master Mix to each tube containing 20 ul first strand cDNA product from Section IV E Mix well and briefly spin in a minicentrifuge to collect the contents at the bottom of the tube s IMPORTANT Transfer the samples from the PCR Clean Work Station to the general lab All downstream processes should be performed in the general lab 4 Place the tube s in a preheated thermal cycler with a heated lid and run the following program 95 C 1 min X cycles 2 98 10sec 65 C 30sec 68 C 3 min 72 C 10min 4T forever aConsult Table 2 for PCR cycle number guidelines STOPPING POINT The tubes may be stored at 4 C overnight www clontech com P
6. A E S EE 18 Appendix B PCR Optimization secnssiiniieceneininciien an a e a a e aa ai 19 Table of Figures Pistire Protocol OVervie wi sissicpscessutcsesesbentecseadeneasslessssdbieesssSsteepuaapostassa NEEE EE OIT EEEE EAE ENE RENEE EER E ERE 3 Figure 2 Flowchart of SMAR Ter CDNA Synthesis es esecsseessecesecesecesecesecsseeseeessneseneeeaeeesaecaaecaaecsaecaecsaeseseeeeeseeeenneesaee 4 Figure 3 SMART adapter in Primer 2 Read ceesseecsseesseceseceencesececaesecesecaesoeessceseeseseesseeessesneeneecateccnseneecoeesseeseeeseeses 5 Figure 4 Electropherogram example results from Agilent 2100 Bioanal yzer eee cesceseceseceseceeeeeseeeeeeeeaeeeaeesaeeeaeeenaees 15 Figure 5 Constructing a magnetic separation device for 0 2 ml tubes from rare earth magnets 0 0 0 cece eeeeceteceeeeeeeees 18 Table of Tables Table 1 Sample Preparation Guideline Soricei iee i a a E a R Ea EN EEN Ea ENE EEN EERE 10 Table 2 Cycling Guidelines Based on Amount of Starting Material 0 0 0 cece ceeceeeceeeceeeeeeeeeseeeeaeeeaeesaecaecsaeceaeceaeenseeees 12 Table 3 Process Configuration Panel Setup ee eesesseesseesseeeeeeeecsaecsaecaecssecsseesseeseeseeeseeeesseeeaeesaeesaaecsaecsaecsaeseaeesseenes 16 Table 4 Cycling Guidelines Based on Amount of Amplified cDNA Section V B Step 2 oo eceeseesceesseeseceseceeseeeeeeees 17 120514 www clontech com Page 2 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA K
7. Clontech Laboratories Inc SMARTer Ultra Low Input RNA Kit for sequencing v3 User Manual Cat Nos 634848 634849 634850 634851 634852 634853 120514 Clontech Laboratories Inc A Takara Bio Company 1290 Terra Bella Avenue Mountain View CA 94043 USA U S Technical Support tech clontech com United Asia Pacific Europe Japan States Canada 1 650 919 7300 33 0 1 3904 6880 81 0 77 543 6116 800 662 2566 Page of 19 SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Table of Contents Table of Contents sieve ccsvessscasseisestavesseceusces ccefedascacovassadavt actu EE EEEE EARE EEEE EE E EE e Ea E E a E 2 Ts ntrod cto epi E E E E E EE E E E EES 3 U Listof Components mrserieie ineneting e ESEE E EA OESE vies vi AE EAE EE EERE E EENES 6 UI Additional Materials Regtired sioscoun ainai eiea E RE E Eana e eivai aeS 7 IV SMAR Tef CDNAS ymthesis cis ccssoccgsceci seins iene a E A E E EE E EE EEE EES 8 A Requirements for Preventing Contamination eee esecssecssecssecesecssecseceseesseeeeneesaeeeaeecaaecsaecsaecaecsaeesseeseeeeeeeseeeesaee 8 Bz General Requite ment cis tssscescasctessascusessslgadtanstovsasesnededrsscucestharaliaredsccdunansatessassucesetlyateualesnexsuasetedessensessuagadtesotecetivastes 8 C Sample Recommendations oi scise cesses giecsseassiedendiecdetiaveigesciesctesedcdonsiadlovs ageitea shee a a a a aas 9 D Sainple Requirement cscs issecsesedeccvtssivse Pah cuaswos siii usin tac
8. PCR 8 tube strip USA Scientific Cat No 1402 4700 Nuclease free low adhesion 1 5 ml tubes USA Scientific Cat No 1415 2600 or LoBind tubes Eppendorf Cat No 022431021 For SPRI Solid Phase Reversible Immobilization Bead Purifications Agencourt AMPure XP PCR purification kit 5 ml Beckman Coulter Part No A63880 60 ml Beckman Coulter Part No A63881 Use this kit for the amplified cDNA purifications Section V A Molecular biology grade 100 ethanol Magnetic separation device for small volumes See Appendix A Use this magnetic stand for the amplified cDNA purifications Section V A Optional depending on the choice of magnetic stand for amplified cDNA purifications Section V A Magnetic Stand 96 Life Technologies Part No AM10027 96 well V bottom Plate 500 ul VWR Cat No 47743 996 MicroAmp Clean Adhesive Seal Life Technologies Part No 4306311 Low speed benchtop centrifuge for spinning a 96 well plate For Sequencing Library Generation The components you need are dictated by which library preparation protocol you follow Section VI Ion Xpress Plus Fragment Library Kit Life Technologies Cat No 4471269 for enzymatic shearing Nextera XT DNA Sample Preparation Kit Illumina Cat Nos FC 131 1024 FC 131 1096 Covaris Instrument and related materials for DNA shearing Low Input Library Prep Kit Cat No 634947 120514 www clontech com Page 7 of 19 Clontech Laboratories Inc A Tak
9. PCR strip tubes One can place strip tubes in a column row of a magnetic separation device designed for use with 96 well plates or use a plate and a 96 well magnet The latter can be useful with a large number of samples Alternatively one can construct a suitable low cost separation device from common laboratory materials Please visit www clontech com rna seq tips for a video tutorial on how to construct your own efficient magnetic separation device Example 1 Using a 96 well Axygen V bottom plate and Life Technologies Magnetic Stand 96 You may use a 96 well Axygen V bottom plate in combination with the Life Technologies Magnetic Stand 96 to purify your PCR amplified cDNA Below are modifications to the protocol described in Section V A Before purification Section V A Step 1 cover all the wells of a 96 well Axygen V bottom plate with a MicroAmp Clean Adhesive Seal You may use a razor blade or scalpel to score the seal and uncover only the wells that you want to use Add AMPure XP beads to the wells and then transfer the entire PCR product to the wells containing beads In order to magnetically pellet the beads place the 96 well plate on the Life Technologies Magnetic Stand 96 Example 2 Building a magnetic separation device from rare earth bar magnets and a tip rack to accommodate 0 2 mi tubes 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 inv
10. 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 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 120514 www clontech com Page 8 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual C Sample Recommendations e Total RNA Extraction The sequence complexity and the average length of SMARTer cDNA are noticeably dependent on the quality of starting RNA material Due to the limiting sample size most traditional RNA isolation methods may not be applicable There are several commercially avai
11. The primer used for amplification of the double stranded cDNA is blocked See Figure 2 which prevents ligation of the sequencing adapter at the 5 ends of the double stranded cDNA fragments containing the SMART sequence In many library preparation methods for Illumina the double stranded adapters are added to the cDNA fragments through ligation Unfortunately in these reactions ligation may also take place between the bottom strand of the cDNA fragment and the Illumina adapter containing Read Primer 2 at a low and somewhat variable rate If ligation is also successful on the other unblocked side of the same cDNA fragment this bottom strand can be amplified by the subsequent PCR and can ultimately form clusters for sequencing on Illumina machines Upon sequencing these clusters the SMART adapter will be present in the first 30 cycles in Read 2 of an Illumina sequencing run In addition the dT30 sequence from the 3 SMART CDS Primer II A will also be present after the adapter in a subset of these clusters The presence of the SMART adapter in Read 2 commonly occurs at a high enough rate to be observed in the base distribution by cycle graph generated by the run analysis Figure 3 cycles 77 106 as does the dT30 sequence Figure 3 cycles 107 136 If you wish to avoid sequencing the SMART adapter there are three options 1 Use the Low Input Library Prep Kit Cat No 634947 This unique adapter addition method does not allow for e
12. Tris Cl pH 8 5 1 7 ml 2x1 7 ml 6 8 ml 2x 6 8 ml 4x 6 8 ml 10 x 6 8 ml 10X Afal Buffer 24 ul 48 ul 96 ul 192 ul 2x 192 ul 5 x 192 ul 0 1 BSA 24 ul 48 ul 96 ul 192 ul 2x 192 ul 5 x 192 ul Afal 10 U ul 12 ul 24 ul 48 ul 96 ul 2x 96 ul 5 x 96 ul 120514 www clontech com Page 6 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Storage Conditions Store Control Total RNA and SMARTer IA Oligonucleotide at 70 C Store 10X Lysis Buffer v3 at 20 C Once thawed the buffer can be stored at 4 C Store Elution Buffer at 20 C Once thawed the buffer can be stored at 4 C Store all other reagents at 20 C Additional Materials Required The following reagents and materials are required but not supplied They have been validated to work with this protocol Please do not make any substitutions because you may not obtain the expected results Single channel pipette 10 ul 20 ul and 200 ul Eight channel pipette recommended 20 ul and 200 ul Filter pipette tips 2 ul 20 ul and 200 ul Minicentrifuge for 1 5 ml tubes Minicentrifuge for 0 2 ml tubes or strips For PCR Amplification amp Validation One dedicated thermal cycler used only for first strand synthesis One dedicated thermal cycler used only for double stranded cDNA amplification by PCR High Sensitivity DNA Kit Agilent Cat No 5067 4626 Nuclease free thin wall PCR tubes or strips 0 2 ml
13. age 12 of 19 Clontech Laboratories Inc A Takara Bio Company V SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Amplified cDNA Purification amp Validation A Protocol Purification of Amplified cDNA using the Agencourt AMPure XP Kit PCR amplified cDNA is purified by immobilization onto AMPure XP beads The beads are then washed with 80 ethanol and cDNA is eluted with Elution Buffer NOTES Aliquot AMPure XP beads into 1 5 ml tubes upon receipt in the laboratory Before each use bring beads to room temperature for at least 30 minutes and mix well to disperse Prepare fresh 80 ethanol for each experiment You will need 400 ul per sample You will need a magnetic separation device for 0 2 ml tubes strip tubes or a 96 well plate If you do not have such a device we recommend constructing one using the instructions in Appendix A 1 Add 1 ul of 10X Lysis Buffer v3 to each PCR product from Section IV F Vortex AMPure XP beads until evenly mixed then add 50 ul of Ampure XP Beads to each sample Mix by vortexing or pipetting the entire volume up and down at least 10 times to mix thoroughly gt w N Incubate at room temperature for 8 minutes to let the DNA bind to the beads NOTE The beads are viscous suck the entire volume up and push it out slowly 5 Briefly spin the samples to collect the liquid from the side of the tube or sample well Place the samples on the magnetic separation device for
14. ara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual IV SMARTer cDNA Synthesis NOTE Please read the entire protocol before starting This protocol is optimized for cDNA synthesis from ultra low input amounts of total RNA using Clontech s SMARTer method The protocol is also suitable for cDNA synthesis directly from whole 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 conditions A Requirements for Preventing Contamination Before you set up the experiment make sure you have two physically separated work stations A PCR Clean Work Station for all pre PCR experiments that require clean room conditions e g first strand cDNA synthesis Protocol IV E NOTES The PCR Clean 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 Strictly obey clean room operation rules A second work station located in the general laboratory where you will perform PCR Protocol IV F and measure cDNA concentration Protocol V B B General Requirements The success of your experiment depends on the quality of your input RNA Prior to cDNA synthesis please make sure that your
15. aterline 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 Elution Buffer to the DNA from Section V A Transfer 75 ul of the Elution Buffer DNA mixture into the 100 ul Covaris tube Put the sample tubes into the appropriate location on the Sample holder Set up the process configuration panel as shown in Table 3 Table 3 Process Configuration Panel Setup Peak Power Duty Burst Cycle Time min Mode 175 10 200 5 min Frequency Sweeping Starting with SonoLab version 7 software Peak Incident Power replaced Intensity as a parameter For previous versions of SonoLab set Intensity to 5 4 Save the file and click return to go back to the main page 5 Open the door Place the tube holder with sample tubes on the transducer positioning system 6 Close the door 7 Click START on the main page to run the process 8 After shearing is complete transfer 75 ul of sheared DNA to 1 5 ml tubes 9 Proceed to generate an Illumina Sequencing Library with the Low Input Library Prep Kit Cat No 634947 Dispose of all tubes and pipettes that have been exposed to amplicons in a sealed trash bag 120514 www
16. clontech com Page 10 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual 5 Place the tubes into a preheated thermal cycler and run the following program 72CT 3min 4 forever NOTE Steps 9 10 below are critical for first strand cDNA synthesis and should not be delayed after completing Step 6 It is therefore recommended that you prepare your Master Mix in Step 8 with all components except the enzyme while your tubes are incubating in Step 5 6 When the thermal cycler reaches 4 C remove the tubes from the thermal cycler and put them on ice 7 Preheat the thermal cycler to 42 C 8 Meanwhile prepare enough Master Mix for all the reactions plus 10 of the total reaction mix volume by combining the following reagents in the order shown at room temperature 4ul 5X First Strand Buffer 0 5 ul DTT 100 mM 1 ul dNTP Mix 20 mM 1 ul SMARTer IIA Oligonucleotide 12 uM 0 5 ul RNase Inhibitor 40 U ul 2 ul SMARTScribe Reverse Transcriptase 100 U ul 9 ul Total Volume added per reaction Add the reverse transcriptase to the Master Mix just prior to use making sure to gently mix the reverse transcriptase tube without vortexing before adding it Mix the Master Mix well by gently vortexing and then spin the tube s briefly in a minicentrifuge to collect the contents at the bottom of the tube 9 Add 9 ul of the Master Mix to each reaction tube from Step 6 Mix the
17. contents of the tubes by gently pipetting and spin them briefly to collect the contents at the bottom 10 Place the tubes in a thermal cycler with a heated lid preheated to 42 C Run the following program 42 C 90min 707 10min 4 forever STOPPING POINT The tubes can be stored at 4 C overnight 120514 www clontech com Page 11 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Protocol cDNA Amplification by LD PCR Perform Steps 1 amp 2 in PCR Clean Work Station PCR Primer II A v3 amplifies cDNA from the SMART sequences introduced by 3 SMART CDS Primer II A and the SMARTer II A oligonucleotide IMPORTANT Table 2 provides guidelines for PCR optimization depending on the amount of total RNA or cells used for the first strand cDNA synthesis These guidelines were determined using the Control Mouse Brain Total RNA Typical cycle numbers are provided as a rough guide for working with small amounts of RNA Optimal parameters may vary for different templates different cell types and different thermal cyclers To determine the optimal number of cycles for your sample and conditions we strongly recommend that you perform a range of cycles See Appendix B for PCR optimization suggestions Table 2 Cycling Guidelines Based on Amount of Starting Material Input Amount Input Amount Typical Number of Total RNA of Cells of PCR Cycles 10 ng 1 000 cells 9
18. erted so the tubes can be inserted Panel B lt ss G si _ A bay Samy Sey Aay Oar At tn tm 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 holding an 8 tube strip of 0 2 ml tubes 120514 www clontech com Page 18 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Appendix B PCR Optimization 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 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 double stranded cDNA will remain in the exponential 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 b
19. esiagd hone RE E AAEE ductv EAEE 9 E Protocol Pirst Strand cCONA Synthests 3 2ccccsiags ideo cetteicou seed Ste n n e A ENKE EESE EEE ER 10 F Protocol cDNA Amplification by LD PCR uu eee eeceesceeneeceesaeceaecsaecaeceseeseeseeeseaeseaeecaeeeaeecaaecaaecsaeseaeenaeenaeeees 12 V Amplified CDNA Purification amp Validation cece eesessceesceeseecssecssecsaecssecsseceseesseeeseeeseeeeaeeeaeeeaeecaaecsaecsaeseaeseaeeaeenes 13 A Protocol Purification of Amplified cDNA using the Agencourt Ampure XP Kit ee eeeeeseeesseeneeceseceseceeenseeees 13 B Validation Using the Agilent 2100 BioAnalyZer eee eeeesseessecssecsseceseceseceseeeseeesceeseeseaeecaeeeaeecaaecaaecsaeenaeenaeeaeeees 15 VI Library Preparation for Sequencing on Next Generation Sequencing Platforms 20 0 0 eeeeseeeseeeseeeneeceseceseceseeeseeees 16 A Protocol Illumina Library Preparation Using Nextera DNA Sample Preparation Kits 2 0 0 ccesseeseesseceseceseeeeeeees 16 B Protocol Illumina Library Preparation Using Covaris Shearing and the Low Input Library Prep Kit 16 C Protocol Ion Torrent Sequencing Platforms ee eeeeseeseesssecssecsecesecesecsseeeseeeseeeeeeeeaeeeaeeeaeecaaecaaecaeseaeeeseeeaeeeas 17 VIL References visits cit tiie teen liiite eluate iE A E E A EE E E Ea E E a Sia 18 Appendix A Constructing an Effective Magnetic Separation Device for use with SPRI Bead Separation from a Small Volume Supernatants assonar aa a a e EA Ea E a E AEE TA E O AE AE
20. etter 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 PCR 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 95 C 1 min X cycles 98 10sec 65 C 30sec 68 C 3 min 72 C 10min 4 C forever 2 Perform Purification of Amplified cDNA using the Agencourt Ampure XP Kit 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 4 Determine the optimal number of PCR cycles required for each experimental and control sample We recommend using the lowest PCR cycle number that generates enough material for Ion library construction 5 Apply the optimal number of PCR cycles to your sample material 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 800 424 1350 toll free Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Not
21. he fragmented DNA following the Ion Shear reaction There is no need to end repair the Ion Shear fragmented cDNA See Table 4 for cycling guidelines Table 4 Cycling Guidelines Based on Amount of Amplified cDNA Section V B Step 2 Input Amount of Amplified cDNA Typical Number of PCR Cycles 10 ng 11 1ng 14 120514 www clontech com Page 17 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Vil References 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 Ramskdld D Luo S Wang Y C Li R Deng Q Faridani O R Daniels G A Khrebtukova I Loring J F Laurent L C Schroth G P amp Sandberg R 2012 Full length mRNA Seq from single cell levels of RNA and individual circulating tumor cells Nature Biotechnology 30 8 777 782 Appendix A Constructing an Effective Magnetic Separation Device for use with SPRI Bead Separation from a Small Volume Supernatant Using an optimized magnetic separation device is essential for efficient SPRI bead separation from a small volume supernatant It can be difficult to find strong magnetic separation devices designed specifically to handle small volumes or 0 2 ml
22. ice 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 HiSeq MiSeq and Nextera are registered trademarks or trademarks of Illumina Inc Clontech the Clontech logo SMART SMARTer SMARTScribe and Ultra 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 120514 www clontech com Page 19 of 19 Clontech Laboratories Inc A Takara Bio Company
23. ill show a reduced yield of cDNA relative to those that have never been frozen However it is possible to use this protocol with previously frozen cells The cDNA synthesis protocol has been tested with suspension cells without internal labeling It cannot be used with cells that have undergone fixation 120514 www clontech com Page 9 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual Protocol First Strand cDNA Synthesis Perform in PCR Clean Work Station First strand cDNA synthesis from total RNA or lysed cells is primed by the 3 SMART CDS Primer II A and uses the SMARTer II A Oligonucleotide for template switching at the 5 end of the transcript IMPORTANT To avoid introducing contaminants into your RNA sample the first part of the cDNA synthesis protocol Section E requires the use of a PCR work station ideally in a clean room 1 Thaw all the reagents needed for first strand cDNA synthesis except the enzyme on ice Gently vortex each reagent to mix and spin down briefly Store on ice 2 Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer v3 with the RNase Inhibitor as indicated below scale up as needed 19 ul 10X Lysis Buffer v3 1 ul RNase Inhibitor 20 ul Total Volume Vortex briefly to mix then spin down 3 See Table 1 for guidelines on setting up your control and test samples Prepare each sample 10 ul t
24. it for Sequencing v3 User Manual l Introduction A SMARTer cDNA Synthesis for Sequencing The SMARTer Ultra Low Input RNA Kit for Sequencing v3 Cat Nos 634848 634849 634850 634851 634852 amp 634853 is designed to generate high quality cDNA directly from 1 1 000 cells or 10 pg 10 ng of total RNA in a convenient input volume of 1 9 pl This kit improves on previous generations of SMARTer Ultra Low kits SMARTer Ultra Low RNA Kit for IIlumina Sequencing and SMARTer Ultra Low Input RNA for Illumina Sequencing HV by simplifying the workflow identifying more genes and increasing the representation of GC rich genes This new kit also provides the benefit of generating cDNA that is compatible with both Ion Torrent and Illumina platform specific library preparation kits The protocol described in this user manual has been optimized for cDNA synthesis using the SMARTer Ultra Low Input RNA Kit for Sequencing v3 only If you are using a previous version of the SMARTer Ultra Low kit please refer to its user manual The kit has been designed and validated to prepare cDNA samples for library preparation and sequencing with the following next generation sequencing platforms Ion Torrent Personal Genome Machine PGM Ion Proton Illumina HiSeq and MiSeq The cDNA synthesis protocol can be completed in five hours and the entire library construction protocol can be completed within two working days Figure 1 SMART technology offers
25. lable 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 Input RNA should be free from poly A carrier DNA that will interfere with oligo dT primed cDNA synthesis e Evaluation of RNA Quality 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 Refer to the manufacturer s instructions for information on how to use the Agilent RNA 6000 Pico Kit e Cell Culture Media When working with cultured cells it is important to select a cell culture medium that does not inhibit first strand cDNA synthesis The protocol in this user manual was validated with cells suspended in cell culture grade PBS D Sample Requirements The SMARTer Ultra Low Input RNA Kit for Sequencing v3 works with up to 9 ul of cells or RNA e Total RNA This protocol has been optimized for cDNA synthesis starting from 10 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 e Cells This protocol has been validated to generate cDNA starting from cells Cells that have been frozen prior to starting cDNA synthesis w
26. nd amplification 18 PCR cycles 120514 www clontech com Page 15 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual VI Library Preparation for Sequencing on Next Generation Sequencing Platforms If you are preparing your library for Illumina sequencing as described below please see Section I B of the Introduction SMART Adapter in Illumina Primer 2 Read for more information on how to avoid sequencing the SMART adapter A Protocol Illumina Library Preparation Using Nextera DNA Sample Preparation Kits The full length cDNA output of the SMARTer Ultra Low Input RNA Kit for Sequencing v3 can be processed with the Nextera XT DNA Sample Preparation Kits from Illumina We recommend using an input amount of 100 150 pg amplified DNA in the input volume recommended in the Nextera XT Sample Preparation Guide Follow the rest of the protocol as written B Protocol Illumina Library Preparation Using Covaris Shearing and the Low Input Library Prep Kit 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 deionized water to the water bath The water level in the cooler should be within 3 mm of the FULL w
27. nown as SMART Segq Ramsk6ld et al 2012 was first developed for single cell transcriptome sequencing Since then there has been a push to continue to improve sensitivity and robustness of single cell mRNA seq methods The current SMARTer Ultra Low Input RNA kit for Sequencing v3 is the latest generation of methods that clearly outperforms previously published protocols including SMART seq For more information on SMART technology please visit www clontech com A schematic outline of the technology and workflow is shown in Figure 2 Total RNA or cell s Neen BIND DD ISIS SII ISS SIAN poly A 3 ion 5 3 SMART CDS SMARTer II A Primer Il A Oligonucleotide First strand synthesis and tailing by RT eae Wee neath Va aVa aVa ata a aa atatavatavas oH Template switching and extension by RT 5 blocked PCR Primer IIA v3 Amplify cDNA by LD PCR with blocked PCR Primer Il A v3 Double stranded cDNA ie nl l_y Figure 2 Flowchart of SMARTer cDNA synthesis The SMARTer II A Oligonucleotide 3 SMART CDS Primer II A and PCR Primer II A v3 all contain a stretch of identical sequence 120514 www clontech com Page 4 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual B SMART Adapter in Illumina Primer 2 Read The use of blocked PCR primers is especially useful when preparing cDNA for library construction on next generation sequencing platforms
28. nt s High Sensitivity DNA Kit Agilent Cat No 5067 4626 See the Agilent High Sensitivity DNA Kit User Manual for instructions 2 Compare the results for your samples and controls see Figure 4 to verify whether the sample is suitable for further processing Successful cDNA synthesis and amplification should yield no product in the negative control Figure 4 Panel B and a distinct peak spanning 400 bp to 10 000 bp peaked at 2 000 bp for the positive control RNA sample Figure 4 Panel A yielding approximately 2 10 ng of cDNA depending on the input NOTE For more information on how a trace of a positive reaction control should look and compare to that of a negative control please visit www clontech com rna seg tips 3 Proceed to Library Preparation for Sequencing on Next Generation Sequencing Platforms Section VI A E Positive Control RNA 10 pg Negative Control 1604 1604 1404 1404 el 1204 1004 M EAEE E EE E a 35 i 100 150 200 300 400 500 600 1000 2000 i 10380 35 i 100 150 200 300 400 500 600 i 1000 2000 i 10380 bp Figure 4 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 shows a clean product following cDNA synthesis and amplification 18 PCR cycles Panel B shows no product in the negative control following cDNA synthesis a
29. nts at the bottom of the tube NOTE Do not make a master mix 15 ul Purified ds cDNA from Section V A Step 14 5 ul lon Shear Plus 10X Reaction Buffer 19 yl Nuclease free water 39 ul Total Volume each reaction Add 1 ul of Afal 10 U ul to each tube containing reaction mix Proceed immediately to the next step to add the Ion Shear Plus Enzyme Mix II Add 10 ul Ion Shear Plus Enzyme Mix II to each tube containing reaction mix and Afal Proceed immediately to the next step to mix the enzyme mix with the DNA and buffer The total reaction volume is 50 wl Set a pipettor to a 40 ul volume and mix the reaction by rapidly pipetting up and down 8 10 times NOTE Do not mix by vortexing Avoid creating bubbles Incubate the tube s in a preheated thermal cycler set to 37 C for 25 minutes NOTE The Ion Shear reaction is very sensitive to the quality of the starting sample and operator handling method The reaction time may need to be optimized under your laboratory conditions and for different median fragment sizes Add 5 ul of Ion Shear Stop Buffer immediately after incubation and mix thoroughly by vortexing for at least 5 seconds Store the reaction tube s on ice Note that you may optionally confirm the shear profile of the purified DNA with 1 ul of eluted DNA as described in Step V B 1 Proceed to generate an Ion Torrent sequencing library with the Ion Xpress Plus Fragment Library Preparation User Guide starting with Purify t
30. otal volume in individual 0 2 ml RNase free PCR tubes or in an 8 well strip a If you are working with purified total RNA transfer 1 9 ul to a 0 2 ml RNase free PCR tube Bring the volume to 9 ul with nuclease free water Add 1 ul of 10X Reaction Buffer b If you are working with cells isolate cells in validated media and transfer to a 0 2 ml RNase free PCR tube Bring the volume to 9 ul with nuclease free water Add 1 ul 10X Reaction Buffer Gently vortex or pipette to mix the sample Incubate at room temperature for 5 minutes See Sections IV C and IV D for more information on working with cells Table 1 Sample Preparation Guidelines Components Negative Control Positive Control Test Sample 10X Reaction Buffer 1 ul 1 ul 1 ul Nuclease free water 9 ul 0 8 ul 0 8 ul Diluted Control RNA 1 9 ul Sample 1 9 ul Total Volume 10 ul 10 ul 10 ul The Control RNA is supplied at a concentration of 1 ug ul It 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 4 Place the samples on ice and add 1 ul of 3 SMART CDS Primer II A 12 uM Mix well by gently vortexing and then spin the tube s briefly in a minicentrifuge to collect the contents at the bottom of the tube 10 yl Cell Total RNA in Reaction Buffer from Table 1 1 ul 3 SMART CDS Primer Il A 12 uM 11 ul Total Volume 120514 www
31. rroneous ligation 2 Use the Nextera XT DNA Sample Preparation Kit from Illumina to prepare your library We recommend using an input amount of 100 150 pg amplified DNA 3 Sequence only from Read Primer 1 If you have already sequenced with Read Primer 2 the SMART adapter sequence can be trimmed from reads prior to mapping to your transcriptome BOAHMASXX Lane 8 Both Surfaces ST Ei Read 2 Cycles 77 152 2 Figure 3 SMART adapter in Primer 2 Read The presence of the SMART adapter in Read 2 commonly occurs at a high enough rate to be observed in the base distribution by cycle graph generated by the run analysis cycles 77 106 as does the dT30 sequence cycles 107 136 120514 www clontech com Page 5 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual ll List of Components The SMARTer Ultra Low Input RNA Kit for Sequencing v3 consists of the SMARTer Ultra Low Input RNA Kit for Sequencing v3 Components not sold separately and SeqAmp 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 Ultra Low Input RNA 634848 634849 634850 634851 634852 634853 Kit for Sequencing v3 12 rxns 24 rxns 48 rxns 96
32. unparalleled sensitivity and unbiased amplification of cDNA transcripts and enables 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 critical for transcriptome sequencing and gene expression analysis Start with Total RNA or Cells Section IV C SMARTer First strand cDNA Synthesis Section IV E o lt O Full Length ds cDNA Amplification by LD PCR Section IV F Amplified cDNA Purification amp Validation Sections V A amp V B S Library Preparation Choices for library preparation are discussed in Section VI Figure 1 Protocol overview 120514 www clontech com Page 3 of 19 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA Kit for Sequencing v3 User Manual The SMARTer Ultra Low Input RNA Kit for Sequencing v3 incorporates Clontech s patented SMART Switching Mechanism at 5 End of RNA Template technology This technology uses the template switching activity of reverse transcriptase to enrich for full length cDNAs containing the 5 end of the mRNA and directly add defined PCR adapters to both ends of the first strand cDNA Chenchik et al 1998 SMARTer advances to the core SMART technology have significantly improved cDNA synthesis efficiency when starting with picogram amounts of total RNA An mRNA Seq protocol k
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